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Christine Quinn Weds Longtime Partner in NYC-Themed Wedding
Guests included Mayor Bloomberg, Sen. Schumer and Gov. Cuomo
By Melissa Russo	• Published May 19, 2012 • Updated on May 21, 2012 at 8:54 am
City Council Speaker Christine Quinn wed her longtime partner, Kim Catullo, Saturday evening in a ceremony surrounded by family, friends and politicians.
The 275-person guest list was a who's-who in politics, including Mayor Bloomberg and his girlfriend Diana Taylor; Sens. Kirsten Gillibrand and Charles Schumer; Assemblyman Vito Lopez; Rep. Carolyn Maloney; and openly gay state Sen. Tom Duane, who joked outside that the music was better at same-sex weddings.
Schumer told reporters it was "not a matter of if, but when" same-sex marriage was legalized everywhere.
The wedding, held at the lofty Highline Stages on West 15 Street, had a "Spring in New York" theme, with each table named for one of the couple's favorite city neighborhoods.
The space was decorated with local wildflowers, including some grown at the nearby Highline Park.
Quinn wore a full-length creme-colored gown designed by Carolina Herrera, and Catullo wore a creme-colored silk suit designed for her by Ralph Lauren. Their makeup was done by Bobbi Brown.
The brides were walked down the aisles separately by their fathers, Lawrence Quinn and Anthony Catullo. Quinn walked down to Beyonce's "Ave Maria," and Catullo walked down to Bruce Springsteen's "If I Fall Behind."
The couple met 11 years ago this September, and their history was detailed in a video shown during the reception. Quinn was given Catullo's mother's ring, which was reset with sapphires, the official stone of September.
Same-sex marriage was legalized in New York last June, and at the time, Quinn tearfully explained how the new law would change things for her and Catullo.
"I was never sure this bill would pass," she said then. "Even this morning as I stood in my apartment getting ready, I was so nervous, because I had begun to plan the wedding in my mind. And I thought, 'What if it doesn't happen again,' the disappointment will be so tremendous."
"It's hard to describe the feeling of having the law of your state changed to say ... what you know in your heart is true, that you are a full member of the state, and that your family is as good as any other family," she said.
Gov. Andrew Cuomo worked hard to legalize same-sex marriage, engaging in often contentious debates before signing the bill into law in June.
On Saturday, as he slowly walked past reporters to watch Quinn and Catullo wed, NBC 4 New York asked him if he felt responsible for the day.
"No," he replied, after walking back out. "Only two people are responsible."
Get the latest from NBC 4 New York anytime, anywhere. Follow us on Twitter, Facebook and Google+. Get our apps here and sign up for email newsletters here. Get breaking news delivered right to your phone -- just text NYBREAKING to 639710. For more info, text HELP. To end, text STOP. Message and data rates may apply.


That's how you do a pop album, other girls just can't.
"Good Girl Gone Bad" is quite good, although very calculated. Almost any of the songs except for 1 or 2 could be singles. Rihanna has found her niche, and she is best suited for dance-type tracks rather than ballads such as "Unfaithful".
✔ "Umbrella", "Don't Stop the Music", "Sell Me Candy", "Lemme Get That", "Rehab" and "Question Existing"
Purchasing Good Girl Gone Bad from Amazon helps support Album of the Year. Or consider a donation?


Here are some items, priced between $50-$100 and $100+. Hover over the picture to see the brand and price, and click through for further details and to buy.
For suggestions under $50, go to the Luscious Gift Guide: Kids – part 1.
…as well as our regular KIDS section in the LUSCIOUS SHOP.
This entry was posted in Uncategorized and tagged CHRISTMAS GIFT GUIDE, luscious gift ideas.


Using the sense of humor that draws thousands of women a year to hear her speak, Kathi Lipp shows wives through simple daily action plans how they can bring the fun back into their relationship even amidst their busy schedules.
Although "The Husband Project" is camouflaged as an adorably written and delightfully funny book, in all reality it's a powerful tool to help facilitate a more solid and fulfilling marriage. Lipp's engaging style of writing presents her many suggestions in a plausible and palatable way.
Many times during the reading of this book I actually laughed out loud at Lipp's humor, yet at other times I felt deeply challenged to be a better wife. But through it all I felt like I was sharing a cup of tea with a good friend—no condemnation to be found, only love and encouragement. Because the author is willing to laugh at herself and be honest about her own past mistakes, I found myself open to her suggestions and insights.
The Husband Project is so much more then a "book about marriage." It's a cornucopia of helpful information—everything from crock-pot recipes to informational websites, recommended books to a list of "guy movies." Chock full of fresh ideas and practical insights, the principles found in "The Husband Project" are sure to infuse marriages everywhere with renewed passion and reawakened love.
Question: I'm wondering what motivated you to write this book?
Kathi: Like most women, I noticed that after just a couple of years of marriage my husband and I had gone form over the top romantics to tolerant roommates. So I started thinking and praying about what would truly make my husband feel loved (and maybe even lucky to be married to me.) I made up a list and began practicing on Roger. Some of the ideas (buying him a gift card to his favorite restaurant and surprising him with an impromptu date) were big hits. Others (like opening mystery clues for an all day adventure) were, let's say, less than successful.
After several flops (hey, I thought the guys at his work would think that his lunch sack covered in hearts was adorable…) I finally got desperate. I asked him, "Okay, what would make you feel loved?" (And yes, I felt pretty pathetic to be asking.) After showing him the list, he gave me thumbs up or down on several of the items. I now had a much clearer plan in place. No, cookies in the shapes of bunnies were not necessary. Homemade raw cookie dough, however, was a big thumbs up.
Question: I see that you recommend doing the project with a couple of friends. Why is that?
Kathi: I'm famous for starting projects that never quite see completion. I have about $700 worth of scrap booking materials and drawers filled with beads and fasteners, all taking up a disproportionate amount of space in my home. We won't even begin to discuss the number of new-and-never-opened books I have jammed on my bookshelves (and under my bookshelves, and under my bed…).
We all have projects that we start enthusiastically and never manage to finish. I don't want this happen to women with The Husband Project.
There are several ways to assure that you stick with it for the long haul. Putting your plan on paper (or jotting it by hand in this book) is certainly one way to up your chances of doing all 21 Projects. Prayer is definitely key to persisting through your project when your husband has been, let's say, less than charming that day. But, by far, the biggest determination about whether you complete The Husband Project, or this book gets conveniently lost under your bed, is having a couple of committed accountability partners.
In the company of good friends, your experience with the project will be more fun, more creative, and you'll increase greatly your chances of completion.
Question: It seems that some of the ideas you talk about some women might find a little too "'50's housewife". Make him meals, use encouraging words (even if you don't feel he doesn't deserve it,) and wearing lingerie. How have women reacted to the book?
Kathi: Yes, it is a hard sell for some women when you start to talk about putting your husband's needs at the top of your to-do list. It is very counter culture to talk about putting your man's desires as a priority and I do get push-back. But, I do have to say that when women do the Project together, there is freedom in knowing that a lot of women are doing this really radical thing together and getting great results.
I am the leader of the "shut up" group at our church. We actually renamed ourselves because I kept getting scolded by my 4 year old son that "shut up" it a bad word... which is true in our household. Our new name is S.U.S.U., and acrostic for "shut up, suit up". We have been at the projects for a few weeks now and are having so much fun! The main focus is our attitude and why we are doing these projects instead of what we'll be getting out of it. However, we have reaped the benefits of honoring our most loved on this earth. There have been random flowers, acts of kindness and so many fun little stories that this note would end up rather long. Thanks for taking the time to come to our church, make us laugh and challenge our lives. What a great project.
Now those are some results that are worth putting on some lingerie for!


Being able to read is a hugely important skill and here at Brown Clee, we believe the key to enabling children to learn is a rich, cross-curricular approach with a high focus on reading for pleasure, combined with the consistent teaching of progressively more advanced skills. The children in our schools are enthusiastic readers and are given many opportunities to engage with and discuss a variety of enjoyable and high quality texts in discrete guided reading sessions, English lessons and in other areas of our Creative Curriculum; our children not only learn to read, they read to learn!
Children learn to read through a variety of approaches and we know that teaching and providing opportunities to practise all of them ensures all children are able to learn in the way that works for them. Our children learn how to retrieve information, draw inferences, make deductions, make predictions and learn to analyse and evaluate a wide range of texts. We acknowledge that phonics is highly important and it is taught daily from reception to year one, before transitioning into spelling in year two. Phonics lessons are interactive and lively, ensuring the children have fun and learn at the same time.
We encourage home reading with rewards for children who are reading consistently at home. Reading encourages a wider vocabulary, as children are exposed to words they haven't heard before, as well as feeding their imagination and creating interest and enthusiasm.
At Brown Clee, speaking and listening is a core part of our curriculum, supporting learning in all areas. Discussion between pupils is encouraged throughout all lessons to share ideas and deepen understanding. Ambitious vocabulary is modelled by members of staff so pupils have examples on which to model their own communication. We have high expectations of listening skills which are carefully developed in their school time, frequently checking that children are fully understanding and processing every situation.
EGPS (English grammar, punctuation and spelling) is taught throughout English lessons and other areas of the curriculum when children are writing. A weekly focus is planned for in grammar and punctuation but previous skills are consistently revisited, building up the bank of National Curriculum expectations. Key Stage 2 Spelling is taught through the No Nonsense Spelling series, using the revisit, teach, practise, apply format.


Gothic adaptation of Lady Macbeth of the Mtsensk, about a young bride sold into marriage.
Occasionally there is a film that has such confidence in what it is offering that it knocks the viewer for six. Such is Lady Macbeth, William Oldroyd's startling debut, adapted from Nikolai Leskov's Lady Macbeth of the Mtsensk by playwright Alice Birch. What the film lacks in levity, bar the occasional moment of gallows humour, it makes up for with an impact that lingers long after the closing credits.
Transported to an English countryside setting from the Russian one of the novella, Lady Macbeth tells the story of a young woman sold into a loveless marriage. Trapped in an unfamiliar house, in a hostile, gendered environment, the audience watches as Katherine's acts of rebellion manifest themselves, starting small then slowly increasing in velocity and assurance until the film reaches its shattering climax. To say more would ruin the impact of these events, but it is safe to say that there are at least three moments where not a single member of the audience were able to breathe. As well as offering a thrillingly bleak Victorian noir, the film explores themes of race, gender, class, and sexuality. Within the film's brisk running time much is packed in that can be untangled and explored long after the film has finished.
The male characters within Lady Macbeth are either weak, repugnant or a combination of the two. There is sterling support work from Christopher Fairbank, Cosmo Jarvis, and Paul Hilton but the two performances that stand out are the lead female characters. Naomi Ackie as the loyal maid, Anna, is fascinating. The nuance and subtle range that Ackie brings to this role make her character all the more intriguing, and I would happily sit through the film again just to trace her journey. That being said, this is Katherine's film and Florence Pugh's performance is remarkable. As Katherine transforms and tests the audience's sympathies, Pugh brings a potent energy to the role. The grounded nature of Lady Macbeth is reflected in the intricacies of Pugh's performance and she carries this film. She is a talent who will go far, and this role is her calling card.
On a technical level Lady Macbeth is exemplary. The use of silence in a movie can be remarkably illuminating. With so many iconic scores littering the history of cinema, audiences expect a score to add further insight. The restraint shown here in the use of a score and the long stretches of silence make every word spoken, every sound heard meaningful. The acts of violence are shocking as they so often break the silence with their ferocity. This is coupled by Ari Wegner's majestic cinematography, and there are moments of true beauty in Lady Macbeth that reward the emotionally exhausting experience for the viewer.
The insights that can be gleaned from a first viewing of Lady Macbeth promise a film that will reward those who return to it at a later date. It is an opaque film, one whose troubling impact lingers long after it has finished. Lady Macbeth is a star-making lead role for Pugh and an electric debut for Oldroyd, a must see, exceptionally rewarding film for audiences.
This is one of the best films I've seen this year. It's gripping from start to finish, there's just not even a little bit of fat in the production, every scene, every piece of dialogue is sparse but meaningful and the actors all carry it off with panache. Florence Pugh is magnificent in the lead role as a cool lady psychopath. The direction is flawless. I felt trapped in a Victorian drama with no escape. The camera work is wonderful, grainy and grim in the candlelight of the cold mansion and you could feel the personality of the characters oozing into your pores from the close-ups. It's quite brilliant. Don't miss it.


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GLEN REA SIGNS HIS NEW CONTRACT TO STAY AT LUTON TOWN!
Midfielder agrees new long-term deal to remain a Hatter
Sign in to iFollow Hatters
We are pleased to put supporters' minds at rest and confirm that after returning from a short holiday, Glen Rea has signed a new long-term contract to further his career with Luton Town!
The 25-year-old midfielder has become a huge fans' favourite at Kenilworth Road, often travelling to away games while sidelined through injury to stand alongside them on the terraces.
Having initially joined the club in March 2016 from hometown side Brighton & Hove Albion, Glen is the club's third longest-serving player after Pelly-Ruddock Mpanzu and Dan Potts.
Speaking after putting pen to paper in the Trophy Room (pictured above), he said: "I'm over the moon to get it done. Really there was only one option for me to take, and that was to sign for Luton Town again. It's where I call home and I'm so delighted to be here and commit my future to Luton Town.
"It's been a great four years and I'm a Brighton fan, but I think 100 per cent, Luton's got a space in my heart – a million per cent! I'm just so delighted that it's done and I can crack on now. It's the start of a new journey and we can only look up."
Over the course of his 152 appearances for the club, Glen has captained the team on many occasions and has been instrumental in the promotions from League Two to the Championship, where his influence was never more evident than on his return to regular action in January after battling back from a serious knee injury.
"To secure our Championship survival two weeks ago was unbelievable, but it's where we feel we should be and 100 per cent we are a Championship club," said the former Republic of Ireland U21 international.
"We are not looking backwards any more. We are looking to go forward and I genuinely believe in my heart of hearts that we are going to do well.
"I genuinely believe that something special is going to happen. I really do think that. It was unbelievable staying in the league, but we are a Championship club so let's move forward now."
Manager Nathan Jones has been a massive influence on Rea's career having coached him in Brighton's development squad, and Glen was understandably overjoyed to see the Welshman return in May to oversee the team's successful fight against relegation.
"No-one can deny that he's an unbelievable manager, he's an unbelievable coach, so we are delighted to have him back," said Glen. "He's unreal and I'm happy to sign again for him. You know my relationship with him, it was a no-brainer really."
On the supporters bombarding the club's social media channels over the past few days, making clear their desire for him to sign his new deal, he added: "This whole club is important to me. From the fans, who are great, every single member of staff here is great, every player here is great, the manager is great.
"It's just a great club to be at and if the fans are delighted to see me stay, then I'm delighted to give them that joy or whatever it is they are feeling. I'm just so happy to be here."
And Glen can't wait to see the Kenny packed to the rafters again when it is considered safe for supporters to return.
"It's a crazy time, but I know they were watching at home, and I'm sure there were celebrations going on back at home – but it's been tough not having them at the grounds, including the away games," he said.
"At the Kenny they are unbelievable, but away they are so important too, so it's been tough not having them. Hopefully this coming season, things might change and they'll be allowed back, but it has been sad not having them in the ground.
"They are a great bunch and Luton is just a great place to be. Everything about the club is unbelievable."

redpajama_set_name: RedPajamaCommonCrawl


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Umzimkhulu – gmina w Republice Południowej Afryki, w prowincji KwaZulu-Natal, w dystrykcie Sisonke. Siedzibą administracyjną gminy jest Umzimkhulu.

Przypisy

Gminy w prowincji KwaZulu-Natal

redpajama_set_name: RedPajamaWikipedia


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Joint Statement - Cammell Laird, Unite and GMB
Following constructive discussions between the trade unions and Cammell Laird, facilitated by the Mayor of Liverpool, Joe Anderson, an agreement has been reached to suspend both current industrial action and the notice of redundancy due to be issued on 10th December 2018 to 291 workers in the Birkenhead shipyard.
This agreement provides for a four week breathing space and paves the way for the establishment of a joint taskforce by the Mayor to involve; Unite and the GMB trade unions, Cammell Laird, Merseyside local authorities and central government departments alongside Cammell Laird customers.
The agreement recognises the need for the yard to remain competitive in bidding for new and future work and all parties agree to work together to ensure this remains the case. The task-force will meet for the first time on Friday 7th December in an effort to formulate an action plan that secures the future for the yard and its workforce.
All parties agree that Cammell Laird has a positive future and that short term measures are required to address immediate problems of work flow into the yard.
The task-force will examine every opportunity to bring forward work scheduled for later in 2019 while seeking new work orders and financial assistance for a major up-skilling programme to ensure the skills necessary to compete in a highly competitive market are available from within the local community.
All parties commit to working constructively and positively during this period to bring about a long-term solution to the current difficulties experienced and remain committed to working together to secure a positive future for a world class shipyard employing a highly skilled workforce from within the local community.
Whirlpool Bristol strike off after firm agrees to workers' demand for £10 an hour minimum
Whirlpool in a spin as Bristol workforce strikes over poverty pay


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Apr 13, 2018 I Nick Redfern
The Strange Saga of Edward Teller and Area 51
Bob Lazar
Edward Teller
Taking into consideration the fact that the saga of Bob Lazar is coming up to its 30th anniversary, I thought I would turn my thoughts into a 3-part feature (here's part-1 and here's part-2). The third and final part revolves around none other than an encounter that Lazar had in the early eighties with a world-famous scientist. It was in 1982 that the Los Alamos Monitor newspaper ran an article on Lazar, which revealed (a) Lazar's love of jet-cars and (b) his then-employment at the Los Alamos Meson Physics Facility. Today, it's the Los Alamos Neutron Science Center.
Just a couple of months after the Los Alamos Monitor ran its feature on Lazar, the man of the hour had a brief encounter with one of the most legendary figures in the world of physics, and someone who became known as "the father of the hydrogen bomb." That man was Edward Teller. When Teller died in 2003 at the age of ninety-five, the U.K.'s Telegraph newspaper noted the following: "A man of enormous intellect, and one of the most controversial scientific figures of the 20th century, Teller made important contributions to the field of quantum mechanics and physical chemistry as well as nuclear physics; but it was as an ardent 'Cold War Warrior' that he entered the popular mind."
On the day on which the two men met, Lazar sat in on a lecture that Teller gave at Los Alamos. It was not so much Teller's lecture that amazed Lazar. Rather, it was the fact that when Lazar was hanging around at the entrance to the facility, there was Teller, sitting on a wall and reading the very article that the Los Alamos Monitor had written on Lazar – which just happened to be a front-page article. Lazar instantly recognized what it was that Teller was so fixated on. So, Lazar decided to make the plunge: he walked over to Teller and told him who he was – namely, the person profiled in the article because of his jet-car achievements. Teller found it all very interesting. The two talked for a while about their respective work, after which Teller headed off inside to deliver his presentation. Six years later, the paths of Lazar and Teller crossed again. It would lead Lazar into the world of Area 51, UFOs and aliens. Or, into a world of disinformation and mind-manipulation.
In 1988, Lazar had a very different job to all of those which came before him. He was living and working in Sin City itself, Las Vegas, where he ran a photo-processing store. It was a job and it paid the bills. It was not, however, the dream job that Lazar wanted. That dream, however, would soon come true. But, it may have also become a definitive nightmare. Lazar decided to send out a resume to just about anyone and everyone he had worked with, met, and knew. One of those – no surprise - was Edward Teller. It's also not surprising that Teller remembered all too well the man with the liquid propane car that could zoom across the landscape at around two hundred miles per hour. Teller also remembered that Lazar had a background in physics. More importantly, Teller was someone who had power, influence, the ability to open doors, and an ability to have access to some of the U.S. Government's most prized and guarded secrets. Almost certainly, Teller played a role in what happened next: namely, that Lazar soon found himself at Area 51.
When Lazar went public with his story of working on alien spaceships at Area 51, he mentioned the Teller connection. It caused more than a few ripples. It's curious that Teller didn't back – maybe even with a lawsuit. This scenario makes even more sense, because Lazar didn't just keep the Teller story to himself. This would have given Teller even more reason – and ammunition – to attack Lazar, verbally and from a legal perspective too. But, Teller did not dispatch a team of high-powered and high-paid attorneys to hammer Lazar into the ground. No. Teller did something very different. Teller made a statement that was so couched in careful terms that it almost became laughable. In fact, it sounds like the carefully chosen words that a lawyer would advise his or her client to use. Teller said, and I quote exactly: "I probably met him. I might have said to somebody I met him and I liked him, after I met him, and if I liked him. But, I don't remember him."
All of this sounds very much like Teller wanting the story to go away, and to go away as quickly as conceivably possible, and in a way that didn't implicate him in any fashion at all. That Teller claimed not to have remembered Lazar is at significant odds with his, Teller's, recall of Lazar in 1988 – six years after the pair had a person-to-person chat about Lazar's passion for super-fast jet-cars. Teller clearly remembered that short chat more than half a decade after it happened. But, no time after the Teller angle became public information in 1989, Teller's memory is suddenly hazy. Very hazy. Or, as today's politicians are so keen to say under awkward circumstances, "I don't recall." Teller hemmed and hawed in what was an embarrassingly awkward fashion.
Did Teller know of the program which saw Lazar manipulated and mind-controlled to ultimately and unknowingly promote a bogus tale of UFOs at Area 51? Whatever the answer, there seems to be little doubt that when the story went public, Teller did just about everything he could to distance himself from Bob Lazar.
Human Beings Are On the Verge of Finally Developing Telepathy
Australian Orbs, Loveland Frogmen, Da Vinci's Paradox Solved, Time Traveler from 2869 and More Mysterious News Briefly
glowing orb
mysterious boom
AI image generator
Jan 03, 2023 I Nick Redfern
Mysterious Deaths: Murders in Bizarre and Weird Ways


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A Better Man (and strong) - 1/1 - Haven fic
Dec. 6th, 2012 at 8:06 AM
Title: A Better Man (and strong)
Chapters: 1/1
Pairing: Nathan Wuornos/Sarah Vernon
Spoilers: The whole series is fair game, but especially the episode Sarah.
Summary: He's a good man, a strong man, a better man than this. What went through Nathan's mind in the moment on the beach?
Disclaimer: I do not own Haven or the characters therein; I can only wish I was that clever and creative! :D
He'd actually managed to take a step away.
And then her voice, soft and mournful, "Why do I always go for the shy ones?" and he froze as a million thoughts sped through his mind.
She wasn't Audrey; he was with Jordan; he was in 1955 for Christ's sake, and if kissing Sarah Vernon wasn't doing something that would impact the space-time continuum, he didn't know what was. But she was vulnerable, and deeply alone in the core of her, just like Audrey (like Lucy? like all the incarnations before Sarah?) and Nathan wanted her to know he understood, that he wanted to do whatever was necessary to soothe that loneliness.
Except he had to get back to 2010 without damaging his reality - or what Audrey said was his reality - and kissing Sarah would not help that. At all.
Only...she wasn't just Sarah Vernon. She was, beneath the surface, the strong, compassionate, funny woman he knew - he knew her, somehow, in the heart of him; and no matter what she was called or the memories she had, he knew the woman underneath it all, and loved her just the same.
He turned to face her, to give Sarah some explanation. He didn't want to leave her feeling like he'd rejected her; like he didn't (love) want her. He simply couldn't do this to her because he had to go back to 2010, and leave her behind in 1955. He could resist her, because he had to resist her. It wasn't fair.
He met her eyes, and thought he was a good man, a better man, a stronger man than this, but he knew, even before he fell to his knees beside her, before he cupped her face and kissed her (her, not just Sarah or Lucy or Audrey, but the person she was beneath all of them), that he was lying.
a better man and strong,
haven,
nathan/audrey,


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InFurNation
Your Guide to the World of Furry Fandom
You've Heard Of Them Before
by rodney.
Of late IDW have become the new home for any number of well-known comic book properties, furry and otherwise. And this includes our old buddies the Teenage Mutant Ninja Turtles. Well now IDW have offered up a little bit of when the Turtles were housed at another publisher — and had some rather popular adventures. Look for Teenage Mutant Ninja Turtles: Urban Legends, Volume 1. "This is it, fans! You demanded it—the entire Image Comics TMNT run (a.k.a. TMNT Volume 3) reproduced for the first time ever in full, four-color glory! Join creators Gary Carlson and Frank Fosco as they take the Heroes in a Half-Shell on some of their most amazing, dangerous, and bizarre adventures ever… culminating in three brand-new issues by Carlson and Fosco to properly close out this long-beloved storyline at last!" Check it out over at IDW's web site.
image c. 2019 IDW Publishing
35 Years of Turtle Power
Hey! We're easing our way into the Christmas Season… How 'bout a gift for the Ninja Turtle fan in your life, courtesy of IDW: The Teenage Mutant Ninja Turtles 35th Anniversary Box Set. "Revisit the glorious origins of the TMNT during the 35th anniversary month! This special box set includes Mirage Publishing's TMNT #1-4 and the Raphael Micro-Series at their original 8 ⅜ x 10 ⅞ dimensions enclosed in a box with new art from TMNT co-creator Kevin Eastman. A must-have for all shell-heads!" You said it, dude.
image c. 2019 Mirage
Mutated and Upgraded
Looks as if next month IDW will release a new Teenage Mutant Ninja Turtles collection of particular note. According to them, it's like this: "Go back to the very beginning, with the Turtles' first encounter with the Foot Clan and their mysterious leader, the Shredder. With over 300 pages of mutated martial-arts action, this volume is perfect for fans to relive the glorious days of the Turtles' origins, as well as an excellent place for new readers to see where the TMNT phenomenon began. Collects the first seven issues of the series, plus the Raphael and Michelangelo Micro-Series one-shots, all in brilliant color." Yep, the Teenage Mutant Ninja Turtles Color Classics Volume 1 was written by Kevin Eastman and illustrated (and colored!) by Peter Laird.
Turtles… In… SPACE!
Yes, obvious, we know, but too good to pass up. Teenage Mutant Ninja Turtles: Dimension X. We'll let IDW explain it: "The brothers make an interstellar journey to different planets in Dimension X to save key witnesses in the Trial of Krang from assassination! Weird new characters, places, and events abound! This mini-series introduces an all-new villain, Hakk-R, into the world of the TMNT, directly affecting the events of the main ongoing series in this dimension-altering adventure." The army of creators for this new mini-series includes writers Paul Allor, Devin Grayson, Ulises Farinas, Erick Freitas, Ryan Ferrier, and Aubrey Sitterson; with art by Pablo Tunica, Michal Dialynas, Khary Randolph, Chris Johnson, and Craig Rousseau. The comic mini-series is out already, and the trade paperback collection is coming later in January.
The Return of the Original Original Turtles
IDW Publishing have gotten really serious with their Artisan Edition line of comic book reprintings. The latest beneficiary? The Teenage Mutant Ninja Turtles, as originally created (of course) by Kevin Eastman and Peter Laird. According to IDW, "This book reproduces all the original art to the first issue of one of the most important comic book phenomenons of the last 35 years — The Teenage Mutant Ninja Turtles. Scanned directly from the original art, mimicking the experience of the original hand drawn page, and also collecting all the layouts (also hand drawn) by the creators of what has been called this generation's Mickey Mouse and Donald Duck. Rounding out the volume is an extensive gallery of other TMNT images, also scanned directly from the original art." Look for Teenage Mutant Ninja Turtles: Artisan Edition, hitting the shelves this May.
When the Bat met the Reptiles. Again.
The first time the Caped Crusader and the Heroes in a Half Shell joined forces to save both New York and Gotham City, it was epic. That time, the story was based on the characters' respective comic books. Now, IDW and DC Comics have hooked up once again to bring us the new Batman/TMNT Adventures 6-issue miniseries — this time, based on the current Teenage Mutant Ninja Turtles CGI TV series and the famous (and influential) Batman: The Animated Series. Written by Matthew K. Manning and illustrated by Jon Sommariva, the first full-color issue hits the stands later this month. Oh, and check out Comics Alliance: They have an extensive write-up on it too.
My Man!
Much of the discussion about the new Teenage Mutant Ninja Turtles movie (Out of the Shadows) has centered on what a welcome addition to the film Bebop and Rocksteady have been. They have always been fan-favorite characters after all. Well now, for the month of June IDW have given the boar and rhino their own comic book line — and a time machine. Be very, very afraid as Bebop and Rocksteady Destroy Everything in a new 5-issue full-color mini-series. According to Comics Alliance, "…the eternal misfits of the TMNT franchise find themselves bouncing around through time itself courtesy of writers Dustin Weaver and Ben Bates, with a cast of artists that includes Nick Pitarra, Sophie Campbell, Giannis Milonogiannis, and Ryan Browne. And if that wasn't enough, they're bringing a few old friends along for the ride, too!" In an unusual (but probably appropriate for these guys) move, IDW is releasing a new issue of the mini-series every week through June — so get yourself caught up!
image c. 2016 IDW Comics
Mutants On The March!
This past spring, the Teenage Mutant Ninja Turtles universe saw the return of one of the famous four's most-popular and perhaps strangest friends: The Mutanimals, appearing in their own five-issue full-color mini-series by Paul Allor, Andy Kuhn, and Ben Bates. Well now IDW are about to release the series as a Mutanimals trade paperback collection. "Old Hob, Slash, Herman the Hermit Crab, Mondo Gecko, and Pigeon Pete have become a force to be reckoned with. When someone close to the group is kidnapped, the Mutanimals end up on an adventure more wild and dangerous than they could have imagined! What mysteries lurk at…The Null Group?" Look for it on the shelves August 19th, or you can pre-order it over at Things From Another World.
Mutagen Gets Around
Another blast from the past over at IDW Publishing as early next year they present the return of The Mighty Mutanimals. From Comicbook.com: "The Teenage Mutant Ninja Turtles' uneasy allies, the Mutanimals, are getting their own minseries from IDW Publishing in February 2015. When someone close to the Mutanimals goes missing, the team consisting of Old Hob, Slash, Mondo Gecko, Herman the Hermit Crab, and Pigeon Pete will be put to the ultimate test, as they try to take on a mysterious new entity known as The Null Group. The series will written by Paul Allor, who has written several TMNT Micro-Seres one-shots, as well as The Ultrom Empire and Turtles in Time miniseries. Andy Kuhn, who created the new IDW design for Mutanimals leader Slash, will provide covers and interior art." In full color of course.
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As I Want
Samaher Alqadi
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TYPE: Feature
Director: Samaher Alqadi
Producer(s): Serge Gordey (Co-Producer, France), Nefise Özkal Lorentzen (Co-Producer, Norway), Jorgen Lorentzen (Co-Producer, Norway), Mohanad Yacoubi (Co-Producer, Palestine)
Key Crew: Gladys Joujou, Senior Editor
ABOUT FISCAL SPONSORSHIP
Fiscal sponsorship is the largest component of the Production Assistance Program. It's designed for women identified filmmakers who are actively fundraising for their film and need 501(c)(3) non-profit tax-exempt status, which is required by many foundations, corporations and government agencies in order to be eligible for funding. Additionally, 501(c)(3) status allows you to receive charitable deductible contributions from individuals—a major incentive for individuals considering making a donation to your project.
An auteur-driven, documentation-of-self, the film depicts an Arab woman's personal process of self-emancipation in a revolutionary struggle against the odds.
The story begins in Cairo, January 25, 2013, the second anniversary of the Revolution, under then-president Mohamed Morsi. Egypt is in turmoil as protests sweep the country against Muslim-Brotherhood rule. An explosion of politically charged sexual assaults against women sweeps through Tahrir Square. After the public rape of her best friend and over 50 other women that night alone, the Director begins to use her camera as a protector and weapon against sexual terrorism as she documents the chaos around her. But she also turns the camera onto herself, to see herself and question what being a woman actually means. In reaction to the shocking events, Egyptian women begin to mobilize and express their rage. Through street marches and women's vigils, she discovers determined women fighting for dignity and their basic survival. As she becomes pregnant mid-way through filming, she re-examines the constructs of her own childhood in Palestine –how her fate was determined because she was born a girl. She initiates an inner-dialog with her deceased mother telling her all the things she was never able to say before. This initiates a visit back to her father's house in Ramallah, where a flood of memories emerges like a forgotten trauma. A series of haunting memory sequences link family archival video, recreations, and the present together. Returning to the evolving social drama playing out on the streets of Cairo, the audience better understands that in each individual woman exists a world of oppression and an eternal struggle for freedom.
Director Statement
Growing up as a woman in Palestine, I struggled severely to gain my independence over the expectations of my family. After I got my first period, I wasn't allowed to play outside with the neighborhood children anymore. I was forced to stay in the house to begin learning how to be a good wife -cooking, cleaning and housework. This film is an examination into the gender inequality Arab society suffers from. I set off to examine my roots and confront painful childhood memories in Palestine to discover what I managed to escape from.
By exploring my own up-bringing I realize how engrained the culture of controlling women is in Middle Eastern society. But it is an outdated social contract. Women are the mothers of this Earth and mothers of all children –as such we demand our respect.
I discovered that Egyptian women decided to raise their heads up and protect themselves by themselves on their own terms. This inspired me to also speak up and show the power of woman. Women did not have the courage before the Revolution to talk about their plight openly, but now it starts to change. Before, these were taboo subjects never discussed in society –a topic that is better left unspoken. It is my hope that by doing so through this documentary film, I will be able to break the silence and open a wider debate that can encourage women to show their power, demand their rights, and change their lives for the better.
- Samaher Alqadi
Supporter Statement
Sørfond Jury Statement (2019): "As I Want" depicts the story of a brutal struggle, of its victims, its villains and its heroines. The script intertwines the dramatic events during the 2013 women´s revolt in Egypt and a personal tale of life as a woman in Palestine.
The jury found this project to be a solid portrayal of women's oppression in a hetero-patriarchal society. A highly relevant and important film for the ongoing struggle of gender equality.
ABOUT FILMMAKER(S)
Director	Samaher Alqadi
Samaher Alqadi is an emerging voice in Arabic documentary filmmaking. Her films focus on the evolving status of women and dissident artists in the Middle East. Raised in Jalazon refugee camp in the occupied West Bank, Samaher studied cinema directing at the High Cinema Institute, Egypt. She Directed and Produced the documentary "Mohammed Bakri" in 2008 about the Palestinian actor and director of the same name who made the controversial film "Jenin Jenin". Samaher's film "Gamalaat" is a short documentary portrait of the legendary Shaabi singer and symbol of matriarchal Egypt Gamalaat Shiha. Her short doc, "Raja3'een" (2012) is a snap-shot of Syrian refugees in France who continue their political struggle in the streets and café's of Paris.
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About GURUREVIEW
GURU REVIEW
UNBIASED REVIEWS OF GURUS
U.G. Krishnamurti
Papaji
George Gurdjieff
Posted on : February 12, 2018 By Editor
George Gurdjieff is one of the brightest figures of contemporary spirituality, and one of the most radical, controversial and contradictory masters ever known. He never gained much popularity, because of the highly innovative and radical character of his teaching, and because he preferred to work with a few chosen disciples rather than having a big following. He was misunderstood and judged by many of his contemporaries, including his own disciples – because he was trying to bring to the western society a completely new type of teaching, that it never knew before.
Gurdjieff was born in Armenia, which was then a part of Russia, from a greek father and armenian mother. The area where he grew up had a great ethnic, cultural and religious diversity. In his childhood he learned a few languages, and by the end of his life he fluently spoke about 10 languages. In his youth he was reading a lot, developed interest in mysticism, and led by curiosity, he went on traveling to find answers for his questions. For many years he was traveling across different countries of Central Asia, Iran, Egypt, India, Tibet, learning from different mystics and mystery schools. He learned to support himself with all kinds of time-jobs and trading schemes, and acquired good business skills. Apparently he was mostly influenced by the practices of sufis (muslim mystics), though he himself never mentioned clearly the sources of his teachings. In 1913 he came back to Russia and attracted his first disciples. From the beginning he was known as an extremely strict and demanding teacher, who was constantly provoking and testing his disciples. After the russian revolution the country became unstable, so Gurdjieff and his group had to change a few places, then they moved to Georgia and finally to Turkey. The next year Gurdjieff went to travel around Europe giving lectures and demonstrations of his work. He unsuccessfully tried to get a british citizenship, and finally settled in France, where he established his Institute for the Harmonious Development of Man. He purchased a small estate south of Paris and stayed there with a diverse group of a few dozen people. His disciples remember that period as a time of spartan life and hard labour, since Girdjieff was practically applying his idea of complex physical, emotional and intellectual development.
In 1924 Gurdjieff had a near-fatal car accident, but against the expectations of the doctors managed to recover. During the recovery he formally dismissed his Institute, but in fact he just got rid of some of his less dedicated disciples. After the recovery he starts writing his main work called "All and everything", according to his own principles he works in the noisy cafes to develop the ability to concentrate. In this period he made numerous trips to the United States, where he already had some disciples, and managed to raise quite a big mount of money for his work. Despite that his estate eventually went into debt and his school was closed. Gurdjieff formed a new group in Paris, all the members were women. After a few years this group was also dismissed.
Gurdjieff continued teaching groups of disciples in his small apartment, even during the World War 2. After the war he managed to reconnect with many of his old disciples, which left him years ago. A year before his death Gurdjieff had another car accident, when he nearly died, but again made an unexpected recovery. Till his last days he was active and even made two trips to the United States.
The contemporaries describe him as a man of iron will, magnetic personality and great physical strength. His behavior was often considered to be immoral, or rather he didn't take morality into consideration at all; for example, he was known for seducing women and had at least 7 unregistered children. He "had a total disregard for the value of mainstream religion, philanthropic work and the value of doing right or wrong in general." The teachings of Gurdjieff are complicated, so is the language his books, which he deliberately wrote in a vague and allegorical manner; he said that reader has to work to find out the meaning. His central idea is that a man as he is lives in a state of sleep, complete unawareness, he doesn't have a unified consciousness, and acts like a machine. Though everyone has an inborn self, that has to be developed through conscious effort, which he called "work". All the religious teachings of the past work through one of the three centers (physical, emotional or intellectual), and therefore are creating an unbalanced individual. His teaching thus was called "The forth way", which unifies three other ways to make the human being complete. The way to get out of a state of daydreaming is to develop awareness or consciousness through different exercises of "self-remembering". One of the main elements of Gurdjieff's work are the Sacred Dances, which are based on some traditional dances and Sufi practices. Each dance has a certain set of movements for different parts of the body, which are performed simultaneously on different rhythmic counts; that requires full concentration and presence during the performance. Each dance has a special music, which was composed by Gurjieff himself. The dances are performed in a group, the participants have to change the position according to a certain scheme, which makes the dances even more complicated. These exercises are meant to develop awareness and continuous presence in action. Gurdjieff was known for creating innovative methods and situations for teaching "on the spot". These could be sudden shouts, abuse, irrational instructions and provocative behavior, which taught the disciple to stay always calm, present and untouched by any situation. Though Gurdjieff's teachings had some influence on the intellectuals of his time, they still remain relatively unknown. There are a few foundations spreading his ideas and many small groups practicing Sacred Dances. His movement remained an enclosed mystery school, which seemingly failed – most of his long-time disciples eventually left him, being unable to stand his unusual methods of work.
Editor	News Reporter
Swami Prabhupada
Meher Baba
Anandamayi ma
Ozen Rajneesh
Osho Rajneesh
Sai Baba of Shirdi
GURUREVIEW 2018 all rights reserved


AHHHH…..MY JAW HURTS!! COULD I BE GRINDING??
Most people probably grind and clench their teeth from time to time, sometimes knowingly and sometimes unconciously. Teeth Grinding, medically called bruxism, does not usually cause harm if it's occasional, but when teeth grinding occurs on a regular basis the teeth can be worn down, significantly damaged and at a higher risk of fracture. Constant pressure on the teeth and constant jaw muscle activity can also result in headaches and pain in the jaw joint, commonly known as the TMJ (Temporomandibular Joint). Often times, patients with TMJ disorders are screened for parafunctional habits of clenching or grinding as their symptoms can be attributed to constant, irregular bite forces created by clenching or grinding.
So why do people grind or clench their teeth????
No specific correlation has been linked to anything in particular but grinding/clenching is usually broughtby stress and anxiety. It can occur while awake, but often occurs during sleep. Although not proven with any solid research, theory suggests that uneven bite and crooked teeth can shift the bite forces on one side of the mouth more than the other causing jaw muscles to fire actively.
How can you find out if you're grinding/clenching????
Because grinding often occurs during sleep, most people are unaware that they grind their teeth. However, a dull, constant headache or sore jaw is usually a good sign. Many times people learn that they grind their teeth by their loved one who hears the grinding at night. Wear patterns on the teeth when examined by dentists is also able to distinguish possibility of grinding.
If you suspect you may be grinding your teeth, talk to your dentist. He or she can examine your mouth and jaw for signs of bruxism, such as jaw tenderness and wear patterns in your teeth including signs of craze lines, fractures or secondary periodontal disease caused by traumatic grinding/clenching.
Constant teeth grinding can result in a generalized sensitivity in teeth, fracturing, loosening, or even loss of teeth. The chronic grinding may wear their teeth down significantly exposing the more sensitive tooth structure of dentin. When these events happen, more invasive restorations such as root canals, crowns, bridges and sometime even a tooth removal may be required.
Not only can severe grinding damage teeth and result in tooth loss, it can also affect your jaws, resulting in the TMJ problems as mentioned earlier. Worn down teeth can cause the facial profile to look much older due to inadequate tooth support for the lips and cheek muscles that surround the mouth.
Lack of adequate tooth height, inappropriate shape and dark color of teeth can cause aesthetic compromises and may cause social awkwardness in individuals.
How can I Stop Grinding?
Your dentist can fit you with a mouth guard to protect your teeth from grinding during sleep or even when you're awake.
If stress is causing you to grind your teeth, ask your doctor or dentist about options to reduce your stress. Attending stress counseling, starting an exercise program like yoga, seeing a physical therapist, or obtaining a prescription for muscle relaxants are among some of the options that may be offered depending on signs and symptoms once examined by your dentist.
Avoid or cut back on foods and drinks that contain acidic content such as colas, sport beverages, excessive juice.
Do not chew on pencils or pens or anything that is not food. Avoid biting your nails. Avoid chewing gum as it allows your jaw muscles to get more used to clenching, making them stronger and making you more likely to grind your teeth.
Train yourself not to clench or grind your teeth. If you notice that you clench or grind during the day, stretch your jaw muscles by finger pressure massage and opening and closing in a normal range five-ten times within a few minutes. This practice trains your jaw muscles to relax.
Relax your jaw muscles at night by holding a warm washcloth against your cheek in front of your earlobe and again stretching your jaw muscles after finger pressure massage.
As always, consult your dental care provider for a proper examination and evaluation. Please call Round Lake Family Dentistry at (847) 740-0217 to schedule your appointment today.


Successful implementation of modified cyanobacteria as hosts for industrial applications requires the development of a cyanobacterial chassis. The cyanobacterium Synechococcus sp. PCC 7002 embodies key attributes for an industrial host, including a fast growth rate and high salt, light, and temperature tolerances. This study addresses key limitations in the advancement of Synechococcus sp. PCC 7002 as an industrial chassis.
Tools for genome integration were developed and characterized, including several putative neutral sites for genome integration. The minimum homology arm length for genome integration in Synechococcus sp. PCC 7002 was determined to be approximately 250 bp. Three fluorescent protein reporters (hGFP, Ypet, and mOrange) were characterized for gene expression, microscopy, and flow cytometry applications in Synechococcus sp. PCC 7002. Of these three proteins, the yellow fluorescent protein (Ypet) had the best optical properties for minimal interference with the native photosynthetic pigments and for detection using standard microscopy and flow cytometry optics. Twenty-five native promoters were characterized as tools for recombinant gene expression in Synechococcus sp. PCC 7002 based on previous RNA-seq results. This characterization included comparisons of protein and mRNA levels as well as expression under both continuous and diurnal light conditions. Promoters A2520 and A2579 were found to have strong expression in Synechococcus sp. PCC 7002 while promoters A1930, A1961, A2531, and A2813 had moderate expression. Promoters A2520 and A2813 showed more than twofold increases in gene expression under light conditions compared to dark, suggesting these promoters may be useful tools for engineering diurnal regulation.
The genome integration, fluorescent protein, and promoter tools developed in this study will help to advance Synechococcus sp. PCC 7002 as a cyanobacterial chassis. The long minimum homology arm length for Synechococcus sp. PCC 7002 genome integration indicates native exonuclease activity or a low efficiency of homologous recombination. Low correlation between transcript and protein levels in Synechococcus sp. PCC 7002 suggests that transcriptomic data are poor selection criteria for promoter tool development. Lastly, the conventional strategy of using promoters from photosynthetic operons as strong promoter tools is debunked, as promoters from hypothetical proteins (A2520 and A2579) were found to have much higher expression levels.
With the current emphasis on renewable energy and sustainable chemical production, cyanobacteria have emerged as photoautotrophic hosts for biofuel and chemical synthesis applications. While metabolic engineering efforts have successfully demonstrated cyanobacterial-based production of a wide range of fuels and chemicals [1–17], cyanobacterial hosts have yet to achieve commercial success. Strain development efforts for cyanobacteria are often limited by the availability of characterized genetic tools and our insufficient understanding of cyanobacterial metabolism and regulation. Complicating these efforts is the fact that multiple, diverse cyanobacterial hosts are used. The most common cyanobacterial hosts for genetic modification include two freshwater hosts, Synechocystis sp. PCC 6803 [5, 9, 10, 15, 16] and Synechococcus elongatus PCC 7942 [2, 4, 12, 18], and a marine host, Synechococcus sp. PCC 7002 [3, 13, 17]. Genetic tools developed for one cyanobacterial host are often directly used in another [13, 17], and cellular processes studied in one cyanobacterium are frequently assumed to be similar, if not identical, in another cyanobacterium. While some tools and cellular processes may be universal to cyanobacteria or eubacteria, this generalization among cyanobacterial species may limit their advancement as industrial hosts. For example, Escherichia coli has been studied and developed as a host for over four decades, and although genetic and functional comparisons may be drawn to other species, recombinant genetic tools have been shown to function in a host-specific manner . In order to achieve advancements similar to that achieved in E. coli, we recommend that metabolic engineers focus on a single cyanobacterial host or chassis.
Synechococcus sp. PCC 7002 is an ideal host for the development of a cyanobacterial chassis. It has a fast doubling time (~2.6 h) . As a marine strain, Synechococcus sp. PCC 7002 growth does not require freshwater resources; this is a key requirement for the production of high quantity, low-value commodities like biofuels. The salt tolerance of Synechococcus sp. PCC 7002 also allows for growth in open raceway pond production systems, where the salt concentration of the growth medium will fluctuate with evaporation. The high temperature tolerance of Synechococcus sp. PCC 7002 also enables growth in photobioreactors, where temperatures often exceed 40 °C . Lastly, modified Synechococcus sp. PCC 7002 has demonstrated enhanced production of free fatty acids compared to the freshwater cyanobacterial host S. elongatus PCC 7942, suggesting improved host tolerance for the production of lipophilic fuels . Despite these advantageous properties, the paucity of genetic tools available for modifying Synechococcus sp. PCC 7002 restricts the advancement of this cyanobacterial chassis.
Foreign genes and even entire pathways are often ported into chassis organisms, requiring either plasmid-based expression or identification of a neutral site for genome integration. As genome integration is more stable and predictable compared to plasmid-based expression, this is often the preferred method for modification, particularly for industrial microbial strains. The desB site in the Synechococcus sp. PCC 7002 genome has historically been used as a 'neutral' integration site, for desB has been shown to function primarily under temperatures much lower than the optimal growth temperature (18 vs 34–38 °C) . However, if Synechococcus sp. PCC 7002 is to be employed under realistic outdoor growth conditions, environmental temperatures are likely to reach the range in which desB is expressed. Therefore, true neutral integration sites are necessary to advance Synechococcus sp. PCC 7002 as a chassis organism. Several putative neutral integration sites have been identified in recent efforts, including the pseudogene glpK (SYNPCC7002_A2842)  and the genomic region between hypothetical protein genes (SYNPCC7002_A0935 and SYNPCC7002_A0936) , yet the neutrality of these sites remains to be verified. Additionally, the annotated pseudogene SYNPCC7002_A2842 was recently shown to be a functional gene; it was originally annotated as a pseudogene due to a frameshift in the DNA sequence that was later shown to be a sequencing error .
Reporters are essential tools for chassis development, as they allow for easy quantitation of gene expression, visualization of subcellular localization, and high throughput screening via fluorescence activated cell sorting (FACS). While fluorescent protein genes and the luxAB bioluminescence operon have been used as reporters for gene expression in Synechococcus sp. PCC 7002 [25–28], there are very few examples of reporters used for microscopy or FACS applications with this organism. Additionally, the influence of cyanobacterial photosynthetic pigments (phycobilisomes and chlorophyll-a) on the optical properties of these reporters has not been characterized. Competitive absorbance of the excitation source, re-absorbance of fluorescent or bioluminescent reporter emission, and signal interference from the photosynthetic pigments may affect the application of these tools in Synechococcus sp. PCC 7002 and other cyanobacteria compared to traditional bacterial hosts.
Lastly, the advancement of Synechococcus sp. PCC 7002 as a cyanobacterial chassis is hindered by the lack of available characterized expression tools. Traditionally, genetic modification efforts in Synechococcus sp. PCC 7002 have relied on a few promoters for expression, such as cyanobacterial promoters associated with photosynthesis or inducible E. coli promoters with poor control in cyanobacterial hosts . Recent efforts from the Pfleger laboratory report the development of promoter tools for Synechococcus sp. PCC 7002 using a random mutant library of the cpc promoter from Synechocystis sp. PCC 6803 and components of the lac and tet repressor systems [26, 28]. While these studies provide orthogonal promoter tools with a wide-range of expression in Synechococcus sp. PCC 7002, very little is known regarding how these heterologous tools will integrate with the natural metabolism and regulation of this organism and how expression may vary across the growth phase and with diurnal cycling. Both circadian rhythm and light conditions have been shown to regulate gene expression in cyanobacteria , and this dynamic regulation will likely be an important design parameter in synthetic biology applications. Thus, tools that can interface with the natural metabolism and regulation of Synechococcus sp. PCC 7002 are lacking.
In this study, we address technical limitations to the advancement of Synechococcus sp. PCC 7002 as a cyanobacterial chassis. Several neutral integration sites were identified and tested for neutrality, and the effect of homology arm length on the efficiency of genome integration in Synechococcus sp. PCC 7002 was characterized. Three fluorescent protein reporters were shown to be useful for microscopy and FACS applications. Twenty-five native promoters from Synechococcus sp. PCC 7002 were cloned upstream of a fluorescent reporter. Characterization of these promoters across the growth cycle and under both continuous and diurnal light conditions allows these promoters to be used as tools for recombinant gene expression and also provides insight into native promoter strength and regulation. This promoter information will provide metabolic engineers with basic guidelines for selecting and designing promoters for use in this cyanobacterial host.
Chemicals were purchased from Sigma-Aldrich (Na2EDTA·2H2O, CaCl2·2H2O, KH2PO4, vitamin B12, ZnCl2, and spectinomycin sulfate), MP Biomedicals (FeCl3·6H2O, CuSO4·5H2O, and CoCl2·6H2O), Acros (Na2MoO4·2H2O), Amresco (NaOAc, 3M, pH 5.2), Fisher Chemical (MnCl2·4H2O and Na2S2O3), and Fisher BioReagents [NaCl, MgSO4·7H2O, KCl, NaNO3, Tris base, H3BO3, kanamycin monosulfate, SDS, chloroform, saturated phenol (pH 4.3), and absolute ethanol]. Enzymes, including Q5 DNA polymerase, Taq polymerase, DNA ligase, and restriction enzymes, were purchased from New England Biolabs. DNA isolations and purifications were performed using the Zyppy Plasmid Miniprep kit, DNA Clean & Concentrator, and Zymoclean Gel DNA Recovery kit from Zymo Research. Genomic DNA was isolated using the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich). All other vendors are indicated in the subsequent methods sections.
Cultures were grown in A+ medium  with antibiotics as needed within a New Brunswick Innova 42R shaking incubator with photosynthetic light bank. The optimum growth temperature for Synechococcus sp. PCC 7002 (34 °C) was used , along with shaking at 150 rpm and an average of 60 µmol photons m−2 s−1 of continuous or 12 h:12 h diurnal illumination from alternating cool white and plant fluorescent lights. Cultures maintained on agar plates were re-streaked every month to maintain the culture, and DMSO (5%) freezer stocks were stored at −80 °C . Cultures were transferred from agar plates to 16 mm glass test tubes containing 4 mL of A+ medium. This test tube culture was grown for 4–7 days and then transferred to 100 mL of A+ medium in baffled 500 mL glass Erlenmeyer flasks with straight-neck flask closure cap at a dilution of 100×. For fluorescence spectra, fluorescence microscopy, and flow cytometry measurements of the strains expressing fluorescent proteins, samples were taken during the linear growth phase (note: when culturing cyanobacteria under these conditions, there is a very short exponential growth phase followed by a linear, light limited growth phase). For the neutral site and promoter expression strains, cell growth and photosynthetic yield or fluorescence measurements were taken every 2 days from the 100 mL cultures. A beaker filled with ultrapure water was maintained within the incubator to minimize evaporative loss of the cultures over time.
where the fraction of PCR positive colonies was determined by re-plating 50 colonies from each transformation plate and screening for insertion of the kanamycin resistance cassette using the screening primers desBscF and desBscR (Additional file 1: Table S1).
All strains used and constructed in this study are listed in Table 1. Putative neutral site (NS) integration strains were constructed using linear PCR fragments for genome integration. The linear fragments include an antibiotic resistance cassette (SpR or KmR) flanked by 500 bp homology sequences from the putative NS. The three fragments were amplified using PCR with Q5 DNA polymerase and stitched together using overlap PCR. The spectinomycin resistance cassette was amplified from pAM2991 (S. Golden, ); the kanamycin resistance cassette was amplified from pSB ; and the homology fragments were amplified from isolated genomic DNA from Synechococcus sp. PCC 7002. Primers used to construct these fragments and the sequences of the linear fragments can be found in the Additional file 1: Table S1. The linear fragments were purified from DNA gels and used for transformation of Synechococcus sp. PCC 7002 as described above.
Genome integration plasmids with varying lengths of homology arms (250, 500, 750, 1000, and 1250 bp) were constructed for integration at desB (Synpcc7002_A0158) in Synechococcus sp. PCC 7002. The knockout plasmid pSB was previously constructed for integration at desB with homology arms of 1000 bp flanking a kanamycin resistance cassette. These homology arms were extended to 1250 bp each and shortened to 750, 500, and 250 bp on each flanking region by amplifying these 5′ and 3′ fragments from Synechococcus sp. PCC 7002 gDNA using Q5 DNA polymerase and the primers listed in the Additional file 1: Table S1. The fragments and pSB were digested with SacI for integration of the 5′ homology arm and with AvrII for integration of the 3′ homology arm. Successful ligation of the 5′ and 3′ homology arms was confirmed using PCR amplification. The resulting plasmids, pSB1250, pSB, pSB750, pSB500, and pSB250, were linearized using SpeI digestion, followed by heat inactivation, and transformed into Synechococcus sp. PCC 7002 as described above.
Three fluorescent proteins, hGFP, Ypet, and mOrange, were selected for expression in Synechococcus sp. PCC 7002. The hybrid GFP (hGFP) sequence includes mutations from both a FACS optimized GFP variant  (Accession Number: U73901), EGFP (Clontech, Accession Number: U55762), and GFPmut2 . All fluorescent protein genes were codon optimized for expression in Synechococcus sp. PCC 7002 using the online codon optimization tool from Integrated DNA Technologies (IDT). The promoter and terminator regions of the native rbc operon flank each of the fluorescent proteins, and the 5′ homology arm (500 bp) from NS2 was placed upstream of the rbc promoter. The entire fragment, containing the 5′ NS2 homology arm (NS2_5′), rbc promoter (Prbc), codon optimized fluorescent protein gene (FP), and rbc terminator (Trbc), was synthesized for each construct using IDT's gBlocks gene fragments. A kanamycin resistance cassette (KmR) and the 3′ homology arm for NS2 (NS2_3′) were amplified using primers KmRF and NS2_3R and inserted downstream of the rbc terminator in each cassette using overlap PCR (see primers in Additional file 1: Table S1). Each PCR amplified linear integration cassette (NS2_5′-Prbc-FP-Trbc-KmR-NS2_3′) was purified and transformed into Synechococcus sp. PCC 7002 as described above.
To construct the NS2 genome integration plasmid with Ypet expression from the rbc promoter, the ypet integration cassette (NS2_5′-Prbc-Ypet-Trbc-KmR-NS2_3′) was amplified using primers to insert SacI and AvrII restriction sites at the 5′ and 3′ ends (Additional file 1: Table S1). This amplified PCR fragment and pSB were digested with SacI and AvrII and ligated to produce pSBPrbcYpet. To allow for exchange of the promoter region, Prbc was removed from pSBPrbcYpet, and KpnI and NdeI restrictions sites were added upstream of ypet to yield pSBYpet (see Additional file 1: Table S1 for primers). For each of the 24 native Synechococcus sp. PCC 7002 loci, 500 bp upstream of the start codon was amplified, digested, and ligated to pSBYpet, yielding plasmids for genome integration at NS2 (Additional file 1: Table S2). The promoter expression plasmids were digested with SpeI and transformed into Synechococcus sp. PCC 7002 as described above.
To estimate cell concentration of the Synechococcus sp. PCC 7002 cultures, optical density (OD) was measured at 730 nm using a PerkinElmer Lambda Bio spectrophotometer. DNA concentration was measured using 2 µL of purified DNA and a Nanodrop 2000 spectrophotometer.
A Jasco J-815 CD spectrometer was used to measure the fluorescence excitation and emission spectra of the strains engineered to express fluorescent proteins. The optimum excitation wavelength for each fluorescent protein was determined from an excitation scan at the optimum emission wavelengths (520 nm for hGFP, 565 nm for Ypet, and 600 nm for mOrange), and the optimum emission wavelength for each fluorescent protein was determined from an emission scan with near-optimum excitation wavelengths (465 nm for hGFP, 485 nm for Ypet, and 515 for mOrange). For each scan, the following settings were used: data pitch = 0.1 nm, sensitivity = 900 volts, Ex bandwidth = 10 nm, Em bandwidth = 10 nm, scanning speed = 100 nm/min, accumulations = 4.
For the promoter expression strains, 200 µL of appropriately diluted culture were placed in a Corning clear bottom 96-well plate, and a BioTek Synergy H4 microplate reader measured optical density at 730 nm. The microplate reader was also used to measure Ypet fluorescence of the promoter expression strains from 200 µL of culture in Costar black bottom 96-well plates with 485/20 nm excitation, 528/20 nm emission detection, a gain of 120, and a read height of 5 mm. Samples that saturated the detector under these conditions were diluted with A+ medium until the fluorescence emission was within the range of detection. Normalized fluorescence readings for each promoter were calculated by using linear interpolation to determine fluorescence readings for culture ODs matching those previously used during acquisition of RNA-seq data  (OD730 = 0.4, 0.7, 1.0, 3.0, and 5.0).
An Olympus IX71 confocal fluorescence microscope with a 60×/1.42 oil objective was used to analyze the fluorescent protein expressing strains of Synechococcus sp. PCC 7002. The culture samples (1.5 mL) were centrifuged at 5000×g for 5 min, and the cell pellets were resuspended in approximately 50 µL of supernatant to concentrate the culture. A 10 µL aliquot of each culture was placed on a glass microscope slide, covered with a no. 1.0 cover slip, and sealed with nail polish. A Prior Scientific Lumen 200PRO fluorescence illumination system with a Sutter Instrument Lambda 10-3 filter wheel was used to excite the samples. The Chroma Chl LP filter cube (Em > 600 nm) with 484 nm excitation was used to detect chlorophyll-a (Chl-a) fluorescence; the Semrock GFP-3035B-OMF-ZERO (Em 520/35 nm) filter cube with 484 or 500 nm excitation was used to detect hGFP and Ypet, respectively; and the Olympus DSU-MRFPHQ (Em 597.5/55 nm) filter cube with 534 nm excitation was used to detect mOrange. SlideBook 6 software was used for image acquisition. The images were imported into ImageJ , upon which Chl-a fluorescence was colored red; fluorescent protein fluorescence was colored green; and scale bars were added.
An Accuri C6 flow cytometer was used for analyzing the Synechococcus sp. PCC 7002 strains engineered with fluorescent proteins. The optimal flowrate for Synechococcus sp. PCC 7002 was determined to be medium speed (35 µL/min, 16 µm core size) based on the best correlation between hemocytometer and flow cytometer cell counts. A cutoff of 50,000 on FSC-H was set, and 20,000 events were recorded for each run. Each sample was diluted with A+ medium so that the number of events per second was less than 650, which was determined to be limit for accurate counting of Synechococcus sp. PCC 7002 cells.
To measure ypet expression levels under 12:12 light:dark conditions, 30 mL samples were extracted from cultures 6 h after the lights turned on and 6 h after the lights turned off after 5 days of incubation under diurnal conditions. The samples were placed in 50 mL ice-chilled, conical tubes and centrifuged at 3900×g for 4 min at 4 °C. The supernatant was decanted, and the cell pellets were immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction. A hot acid phenol extraction method was used for RNA extraction, as described previously for S. elongatus PCC 7942 . Any remaining DNA was removed from the RNA samples using the TURBO DNA-free kit (Ambion, Life Technologies). Isolated RNA was quantified using the Quant-iT RiboGreen RNA assay kit (Life Technologies) with fluorescence measured by a NanoDrop 3300 fluorospectrometer. Complementary DNA (cDNA) was synthesized using approximately 2 µg of RNA and a Superscript III First-Strand synthesis kit with random primers (Invitrogen, Life Technologies). Any remaining RNA was removed using RNase OUT, provided within the cDNA synthesis kit. The cDNA was diluted 10× and used as template with primers (200 µM final concentration) to amplify a 159 bp region within ypet (Additional file 1: Table S1) along with Power SYBR Green PCR Master Mix (Life Technologies) in an Applied Biosystems 7300 Real-Time PCR system for quantification. For relative quantification, rnpA, previously reported as a stable housekeeping gene for qPCR , was used as a reference gene with a 176 bp amplicon (see Additional file 1: Table S1 for primers). Three technical replicates were included for each sample along with no template and no reverse transcriptase controls. The three technical replicate CT values were averaged, and the 2−ΔΔCT method was used for relative quantification . Two biological replicates were analyzed for each promoter expression strain, and the average of these biological replicates is reported along with the standard deviation.
To identify neutral sites (NS) for genome integration in Synechococcus sp. PCC 7002, the genome sequence was analyzed to detect large regions within the genome with no predicted function or annotation. Only three such regions were found to be greater than 1 kb in the Synechococcus sp. PCC 7002 genome: nucleotides 963,217–964,242 between SYNPCC7002_A0932 and SYNPCC7002_A0933 (neutral site 1—NS1), nucleotides 1247,018–1248,056 between SYNPCC7002_A1202 and SYNPCC7002_A1203 (neutral site 2—NS2), and nucleotides 1,864,422–1,865,821 between SYNPCC7002_A1778 and SYNPCC7002_A1779 (neutral site 3—NS3). Genome integration fragments were designed for the first two putative neutral integration sites, using spectinomycin adenyltransferase (aadA), a spectinomycin resistance cassette, and neomycin phosphotransferase (neo), a kanamycin resistance cassette, flanked by 500 bp sequences homologous to NS1 and NS2, respectively. These genome integration fragments were used to construct ΔNS1 and ΔNS2 strains of Synechococcus sp. PCC 7002, as described in the Materials and Methods section. Under standard growth conditions (34 °C, 150 rpm, and 60 µmol photons m−2 s−1 of continuous light), ΔNS1 and ΔNS2 did not show any significant changes in growth or photosynthetic efficiency compared to the wild type (Table 2, two-tail p values >0.3), suggesting that NS1 and NS2 are neutral integration sites under these conditions.
Another important consideration for genome integration is the required length of homology arms for efficient homologous recombination in Synechococcus sp. PCC 7002. Homology arms are the DNA regions homologous to the target site in the genome and flanking a selectable marker. We tested homology arms ranging from 250 bp to 1250 bp for integration of the kanamycin resistance cassette at desB in Synechococcus sp. PCC 7002. It is important to note that these homology arms were cloned into an integration plasmid which was linearized for transformation rather than PCR fragments which would be susceptible to exonuclease degradation. As expected, we found that the transformation efficiency of the integration plasmid increased with increasing length of the homology arms and time of incubation (Fig. 1). Under the conditions used for transformation of Synechococcus sp. PCC 7002 in this study, homology arms of 250 bp appear to be the minimum length required for successful transformation, as only 0–84 colonies were obtained with this integration plasmid across incubation times ranging from 2 to 24 h. The transformation efficiency calculation accounts for the fraction of colonies that have integration of the kanamycin resistance cassette, as confirmed by PCR screening of 50 colonies for each integration plasmid with three biological replicates. Integration plasmids with homology arms greater than 750 bp had a high percentage of colonies with confirmed integration, >86 ± 13%; while integration plasmids with homology arms of 500 and 250 bp had only 61 ± 17 and 34 ± 26% of colonies with positive PCR bands, respectively. Thus, targeted integration of the resistance cassette also increased with increasing length of the homology arms.
Fluorescent protein reporters are often used in chassis organisms to analyze gene expression or to determine the subcellular localization of a recombinant protein by fusing the target protein with the reporter. The use of fluorescent protein reporters in cyanobacteria is complicated by absorbance and fluorescence of native photosynthetic pigments. Therefore, fluorescent protein reporters for Synechococcus sp. PCC 7002 were selected based on the absorbance and fluorescence spectra for this host. Three fluorescent protein genes were codon optimized for expression in Synechococcus sp. PCC 7002: a hybrid green fluorescent protein gene (hgfp), a yellow fluorescent protein gene (ypet), and an orange fluorescent protein gene (morange) . The native rbc promoter and transcription terminator regions were placed upstream and downstream of each codon-optimized fluorescent protein gene; a kanamycin resistance cassette was placed downstream of the fluorescent protein operon; and 500 bp homology arms for genome integration at NS2 were placed as bookends for the entire insertion fragment. The resulting PCR fragments were used to generate engineered strains containing the three fluorescent protein reporters.
The excitation and emission peaks for each Synechococcus sp. PCC 7002 strain expressing a fluorescent protein were determined to be Ex 468–490 nm and Em 507 nm for hGFP, Ex 517 nm and Em 530 nm for Ypet, and Ex 545 nm and Em 560 nm for mOrange (Additional file 1: Figure S1). Using 488 nm excitation, the emission peaks from each of the fluorescent proteins were clearly distinguishable from the spectra of the wild type, but the fluorescence emission peak from hGFP showed some overlap with the shoulder of the fluorescence emission peak from the chlorophyll-a Soret band (Fig. 2). The engineered strains expressing fluorescent proteins were also analyzed using confocal fluorescence microscopy to determine the feasibility of utilizing these reporters for cellular imaging. As shown in Fig. 3, all three fluorescent proteins were visualized along with chlorophyll-a as a control. Lastly, the strains expressing fluorescent proteins were analyzed using flow cytometry to illustrate the feasibility of utilizing these reporters for FACS. Additional file 1: Figure S2 shows that the hGFP and Ypet expressing strains were readily distinguished from wild type using 488 nm excitation and a 533/30 nm emission filter (FL-1) for detection; however, the mOrange expressing strain was not clearly identified by the Accuri C6 flow cytometer's 488 nm excitation and 533/30 nm (FL-1) or 585/40 nm (FL-2) detection filters. A flow cytometer equipped with laser excitation near 548 nm and an emission filter near 562 nm, the excitation and emission maxima of mOrange, should allow for detection and subsequent sorting of mOrange expressing strains.
In order to select a representative sample of native promoters for cloning and characterization, RNA-seq data previously collected for Synechococcus sp. PCC 7002 at various cell concentrations throughout its growth profile (OD730 = 0.4, 0.7, 1.0, 3.0, and 5.0)  were used to select promoters of various strengths (10−2, 10−3, 10−4, and 10−5 counts/total counts) and expression patterns (constitutive, linear phase, and stationary phase). For each expression pattern, two promoters were selected for each strength, yielding 8 promoters for a given expression pattern. Overall, 24 native promoters from the RNA-seq data were analyzed along with the commonly used rbc promoter for comparison (Table 3). This promoter list includes several adjacent gene loci that were predicted by the Database of prOkaryotic OpeRons (DOOR)  to be independent operons containing similar functional protein genes (A1929/A1930 and A1961/A1962). For each promoter, a 500 bp sequence upstream of the target gene was cloned and placed upstream of ypet in pSYpet. After integration of each promoter-ypet fragment into NS2 of Synehococcus sp. PCC 7002, the promoter expression strains were analyzed for Ypet expression under both continuous (60 µmol photons m−2 s−1) and diurnal (12 h/12 h light/dark) conditions.
As the RNA-seq data was collected under continuous light conditions (250 µmol photons m−2 s−1) , the Ypet expression levels measured under continuous light in this study (60 µmol photons m−2 s−1) were used for comparison (Table 3). In general, the expression levels measured by Ypet fluorescence of the engineered strains were low; in fact, only 7 of the 25 promoters showed more than a 50% increase in Ypet fluorescence compared to the wild type. This includes moderate promoters, demonstrating an average 2- to 6-fold increase in Ypet fluorescence (PA1798, PA1930, PA1961, PA2531, and PA2813), and two strong promoters (PA2520 and PA2579) showing an average Ypet fluorescence increase of greater than 30-fold. The moderate promoters drive expression of genes involved in photosynthesis (A1930, A1961), carbon fixation (A1798), cell wall structure (A2813), and a hypothetical protein (A2531) in Synechococcus sp. PCC 7002, while the strong promoters both control hypothetical protein genes (A2520, A2579). Of the two adjacent gene loci analyzed in this study (A1929/A1930 and A1961/A1962), only the most 5′ promoter region showed significant Ypet expression (PA1930 and PA1961) while expression from the downstream loci promoters (PA1929 and PA1962) were indistinguishable from the wild type. Both the expression levels and regulatory patterns were inconsistent between the RNA-seq and Ypet expression data. Some of the highest counts per total counts from the RNA-seq data yielded very low Ypet expression (A0740, A1173, A1181, A2062, and A2210) while the second highest Ypet expression level was observed with a promoter from one of the lowest RNA-seq datapoints (A2520). Additionally, many of the engineered strains showed constitutive patterns of Ypet expression, despite the predicted trends from RNA-seq. This lack of correlation between RNA-seq and Ypet expression data is quantified by correlation coefficients, which were calculated across the growth profile (Table 2). Only one gene locus promoter has a correlation coefficient near one (PA2165), while many other loci promoters show strong negative correlations.
In addition to investigating continuous light conditions, the promoter expression strains were also subjected to diurnal conditions. The only promoter showing a significant change in overall expression levels under diurnal conditions was the rbc promoter, with a 43% decrease compared to continuous light conditions (Fig. 4). For all other promoter expression strains, there was no significant change in normalized Ypet fluorescence between continuous light and diurnal conditions. This is not surprising, as the time scale for degradation of the fluorescent protein signal (half-life of 24 h ) exceeds the length of the diurnal cycle (12 h light, 12 h dark). Therefore, mRNA levels of ypet were measured 6 h after initiation of the light period and 6 h after the start of the dark period for cultures exposed to 5 days of 12:12 diurnal light conditions. The fold-changes in ypet expression between light and dark conditions for each promoter expression strain are shown in Table 4. Only two promoters had more than a twofold increase in ypet expression under light conditions compared to dark conditions (PA2520 and PA2813), and two additional promoters had more than a 1.5-fold increase in expression (PA1731 and PA1181). Of the 25 putative promoters analyzed in this study, only four promoters had reduced expression under light conditions, and the level of decrease in gene expression was less than 35%. The overall average change in gene expression under light conditions for the 25 putative promoters was 1.31 ± 0.34, indicating a slight enhancement of expression in the light.
In an effort to determine the underlying sequences responsible for the expression levels and regulatory patterns observed in this study, we analyzed various groups of the characterized promoter sequences using the online Melina II program , which provides analysis from four different motif finder programs (Consensus, MEME, Gibbs, and MDscan). By analyzing all 25 Synechococcus sp. PCC 7002 promoters cloned in this study, a common motif was identified by all 4 programs with at least one motif identified in 11 of the putative promoters (Additional file 1: Figure S3A). Analysis of promoter subsets grouped by functional classification (strongly expressed, moderately expressed, and diurnally expressed promoters) revealed only one additional promoter motif in the moderately expressed promoters (Additional file 1: Figure S3B). The functional roles of these putative motifs remain to be investigated experimentally.
The DNA transformation process is the first step in constructing a genetically engineered strain, and as such, this process should be optimized for chassis organisms. In this study, we assessed two tools for improving transformation in Synechococcus sp. PCC 7002: neutral integration sites and the homology arm length required for genome integration via homologous recombination. By searching for large gaps in non-coding sequences within the Synechococcus sp. PCC 7002 genome, we identified and experimentally confirmed two putative neutral integration sites. It should be noted, however, that only standard growth conditions were tested in this study, and subsequent use of these integration sites under non-standard conditions must be tested with appropriate controls to confirm their neutrality. The longest homology arms tested in this study (1250 bp) yielded the highest transformation efficiency in Synechococcus sp. PCC 7002; in practice, however, genetic engineers must balance between improved transformation efficiencies and the expense of longer homology arms—either due to the cost of DNA synthesis or the additional time required to clone larger fragments rather than having them synthesized. The minimum length of homology arms was determined to be 250 bp for Synechococcus sp. PCC 7002. Interestingly, this is more than 200 bp longer than the minimum homology arms reported for E. coli [44, 45], suggesting that homologous recombination may be less efficient in Synechococcus sp. PCC 7002 or that native exonuclease activity may degrade the linearized plasmid containing the integration cassette. This homology arm length is similar to the 300 bp length reported previously for both Synechocystis sp. PCC 6803 and S. elongatus PCC 7942 . If the minimum homology arm length for genome integration in Synechococcus sp. PCC 7002 can be reduced, integration cassette construction may become more efficient, either through a lower cost of synthesizing the DNA cassette or the use of PCR primers to provide the homology arms, as reported previously for E. coli . The development of highly efficient DNA transformation and genome integration in Synechococcus sp. PCC 7002 will facilitate strain construction in this host and possibly enable the adoption of high throughput genetic engineering techniques .
Based on fluorescence excitation and emission properties, three fluorescent protein genes were expressed in Synechococcus sp. PCC 7002. We demonstrated the application of these fluorescent proteins as reporters for the quantification of gene expression, as labels for fluorescence microscopy, and as markers for selection via flow cytometry. These techniques may improve our fundamental understanding of Synechococcus sp. PCC 7002 and enable the manipulation of this microorganism for biotechnological applications. A potential limitation in the application of fluorescent proteins in cyanobacteria should be considered, however. The native photosynthetic pigments in cyanobacteria, namely the phycobiliproteins and chlorophyll-a, may (1) compete with the recombinant fluorescent protein for absorption of the incident light for excitation, (2) re-absorb fluorescence emitted by the fluorescent protein, and (3) emit fluorescence that affects the excitation or emission of the engineered fluorescent protein. This optical interference from native pigments should be carefully considered when designing experiments based on recombinant fluorescent proteins and during the interpretation of results. For example, the relatively low expression of Ypet for most promoter expression strains in this study may be influenced, at least in part, by optical interference from the photosynthetic pigments via reabsorption of the emitted Ypet fluorescence. In our laboratory, we have found a significant reduction in fluorescent protein signals in Synechococcus sp. PCC 7002 when compared to common microbial hosts such as E. coli (data not shown), yet a full analysis of transcript and protein levels remains to be conducted to confirm the cause of this reduced fluorescence.
In order to develop promoter tools for Synechococcus sp. PCC 7002 that can interface with the native regulatory system, we analyzed 24 native promoters selected based on their reported expression level and regulatory pattern from previous RNA-seq data collected by the Bryant laboratory . By measuring fluorescence produced from these promoters in modified Synechococcus sp. PCC 7002, we found very poor correlation with the RNA-seq expression data. The discrepancy may be due, in part, to the fact that the fluorescent protein signal has a much longer half-life (>1 day) than the mRNA (<2 min) [48, 49], yet this would only explain an increase in Ypet fluorescence compared to the RNA-seq data. To improve accuracy in expression studies, a degradation tag may be added to the fluorescent protein to reduce the half-life to approximately 1 h, as recently reported in Synechocystis sp. PCC 6803 . The lack of correlation between transcript and protein levels is not particularly surprising, given that similar data has been reported for other organisms . Overall, the results of this study suggest that transcriptomic data should not be the basis for promoter tool development in Synechococcus sp. PCC 7002.
Characterized promoter tools for recombinant gene expression are essential for chassis development. The rbc promoter is commonly used as a strong promoter for expression in cyanobacterial hosts, yet only moderate levels of expression were detected from the rbc promoter in Synechococcus sp. PCC 7002 (Fig. 4). We identified two strong promoters in Synechococcus sp. PCC 7002, PA2520 and PA2579, which demonstrated more than fivefold higher expression than that measured from the rbc promoter. Both of these promoters drive expression of hypothetical protein genes in Synechococcus sp. PCC 7002, which may perform important functions based on their high expression levels. While expression level is the typical metric for characterizing promoters, diurnal regulation is also an important metric for a cyanobacterial chassis. By analyzing mRNA levels of ypet, PA2520 and PA2813 were identified as having more than a twofold increase in expression under light conditions compared to dark, while PA1731 and PA1181 had more moderate, but still significant (>1.5-fold), increases. As such, these promoters may be useful tools for engineering diurnal control of recombinant gene expression. Interestingly, the rbc promoter showed reduced Ypet expression under diurnal light conditions (Fig. 4), yet there was no significant change in the ratio of ypet expression under light vs. dark conditions (Table 4). This suggests that expression from the rbc promoter may be constitutively lower under diurnal conditions or that a finer time resolution of transcript levels is needed to detect diurnal changes. Overall, this study identified and characterized two strong promoters and five moderate promoters as tools for controlling gene expression in Synechococcus sp. PCC 7002. Furthermore, two strong and two moderate promoters were determined to have diurnally regulated expression, with increased expression under light conditions.
Genetic tool development for cyanobacterial chassis is still at an early stage. This study, along with other recent efforts in cyanobacterial tool development [26, 28], aims to advance Synechococcus sp. PCC 7002 as a 'green' chassis, enabling the biological production of useful metabolites from CO2 and sunlight to support industries including biofuels, nutraceuticals, and specialty chemicals. In addition to industrial applications, these genetic tools will help to advance our fundamental understanding of cyanobacteria and their functional role in the Earth's ecosystems.
AMR conceived of the study, constructed all engineered strains, conducted experiments (testing of neutral integration site strains, optimization of homology arm length, fluorescent protein analysis, and promoter expression strain testing), performed data analysis, and drafted the manuscript. TJJ assisted with experiments and analysis for optimization of homology arm length and promoter expression strain testing. LMS contributed to the promoter expression experiments and associated data analysis. All authors read, edited, and approved the final manuscript.
The authors acknowledge Dr. Bryan Carson (Sandia National Laboratories) for his generosity in sharing equipment.
This manuscript does not include data collected from humans or animals.
This work was supported by Laboratory Directed Research and Development funds at Sandia National Laboratories. Sandia is a multi-program laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract DE-ACO4-94AL85000.
12934_2016_584_MOESM1_ESM.docx Additional file 1. Additional information.