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Golomski joins Marian women's basketball coaching staff
Marian University head women's basketball coach Christine Keller has announced the addition of Katie Golomski to the coaching staff as an assistant coach.
"I'm very excited to have Katie on staff," commented Keller. "She has tremendous knowledge of the game, not to mention her experience playing and coaching at the college level. She will be a significant piece to help continue the building our culture."
Golomski joins the Sabre women's basketball program after spending three seasons at Lindenwood University-Belleville as a graduate assistant. During her tenure, the Lynx went on to a combined 55-39 record including a pair of 22-win seasons in her final two seasons. The Lynx qualified for the NAIA National Tournament in those final two seasons but were knocked in the first round both seasons.
"I want to thank coach Keller for giving me the opportunity to be a part of her staff," commented Golomski. "I am excited to continue my coaching career at Marian University."
During her playing career, Golomski was a stand-out at William Penn University connecting on a school-record 268 three-pointers in her career. Her accolades as a Statesman include three honorable mention All-Midwest Collegiate Conference honors along with being named the Midwest Collegiate Conference Newcomer of the Year following her freshman season. Golomski closed out her career 22nd on the all-time scoring list at William Penn with 1,087 points in her career. In the classroom, she was also named to the Midwest Collegiate Conference All-Academic team all four years of her career.
Golomski spent her high school career at Pius XI in Milwaukee, Wis. where she helped lead the 2009-10 squad to a state championship.
–FightBlueFight–
Marian athletics suspends in-person attendance indefinitely
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Epidemic Johto
Welcome to New Johto;
This is what remains of the region after the virus hit.
Undead pokemon lurk behind every corner, infest every city, haunt every cave.
Dark Days are Ahead...
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The SILVER Team (PLOT)
Post n°26
Re: The SILVER Team (PLOT)
by Blue on Wed May 13, 2015 5:09 am
Mount Silver [Base] | Dusk
Blue silently sighed, feeling another pair of eyes on him, he snapped up in time to see Umbreon give him one crimson eyed glance before vanishing into the elongating shadows from the setting sun. Umbreon wasn't angry, was he? Blue did not dare be angry, he remembered the days of when he took it out on his pokemon. Umbreon only wanted what was best, something that the boy himself was unable to see clearly every damn time Daisy and him visited the black haired prick--
Daisy.
The blow was too sharp, too far out of his expected thoughts. Like every time he remembered his clear headed sister, the trainer felt his throat constrict. She was a healer, not a battler. Daisy was no battler... At least that is one thing we have both lost, he thought bitterly, spitting mentally an Red. Blue removed his pack, letting it hit the ground with a hard thump! before snapping around to give a slit eyed glare to the other.
Who the hell did he even think he was? Blue did not dare keep the act of defiance for long, turning away before he pissed himself off. The pain of losing everything, the regret too strong. If he had of stayed, would Lance of gone easy on him? Would Daisy still be alive? His mom? Physically biting his lip, Blue had to make the effort to keep from-- from what? Wailing? Crying? Screaming?
No. No, inside, he knew Red was right. And because of that he could never admit it. Without a word he started pulling out a tightly wound sleeping bad, trying to ignore the presence behind him. But his painful thoughts sobered him, the hardness in his features no longer petty, but defensive and exhausted.
[ time skip? ]
by Red on Fri May 15, 2015 4:07 pm
Mount Silver [Base] | Late Night (39)
Red had watched Blue with his fierce eyes, waiting and anticipating any other attack his rival could have attempted. However, Blue seemed content to simply flop down onto his sleeping roll, his thoughts obviously stewing around in his head-- but not before giving Red one last stern glare, which Red was more than happy to reciprocate.
Blue's optimism and damned near heroism cut Red to the core. He'd been like that once; they both knew it. But with everything that had happened, everything that he'd seen and done... there was nothing left of that boy anymore. No passion, no hope... and it hurt to see Blue as his mirror. That he could have been like that. That maybe if he'd been smart enough, he could have ran and still had his pokemon with him... He could have done so many different things differently...
...but he hadn't.
The stern truth was a dagger in his chest, reminding Red of its existence. He hadn't done any of that. He'd played the good Champion and tried to get Pallet evacuated and then he'd fallen prey to the politics in Indigo Plateau. Drafted by guilt, trained by necessity, forced to murder with mutiny, and then finally, silenced by blackmail. And even though Pikachu managed to make it out alive, who knew whether or not he'd survived YEARS of fighting for his life? For all Red knew, he was also one of those monsters. Maybe he'd been one all along. The thought made his chest involuntarily twitch, but he expertly quashed the clawing sob without a sound. His gaze now blank, he simply let his eyes go to Blastoise's hole to watch the sky darken to a cold, twinkling pool of stars.
Mount Silver (1st floor)|| Late Night
Red didn't sleep. He dozed, napped, rested his eyes, but not sleep. Not real sleep. Never real sleep. Wearily, for what seemed the hundredth time that night, he allowed his eyes to open as the dying sounds of his betrayed pokemon ripped at his mind and soul in what could have been a dream. His breathing was quick but still relatively light and he had begun sweating despite the chill in the damp air. He didn't know if he'd made noises while unconscious or not, but he prayed for his own sanity he'd remained quiet. Taking in a full breath, he closed his eyes as it hitched in his tight throat and he realized that his face was wet. God, had he been crying in his fucking sleep? That's just pathetic...
He shifted, wiping the back of his gloved hand to rid himself of the tears while he tried to alleviate his now numb ass. He probably shouldn't have been sitting up, but whatever.
by Blue on Mon May 18, 2015 10:41 am
The night was long. Blue could not relax, he could not get comfortable, the floor was hard as all hell. But really his mind was making excuses. With Red so nearby, there was no way he could quell all of the mixed feelings inside of him. The anxiety was a compression on the trainer's chest that refused to give up. Blue was all too used to having Arcanine or Umbreon close by, within eyeshot, but though he saw a flash of gold rings sometime during the night, his groggy mind could have been playing tricks on him. His thoughts seeped into his dreams, visions of his sister all the more vivid than before, worse since Red was nearby. But the one that woke him up was an odd one, a nightmare blue had never experienced.
It was what had once haunted him, the pair of dark eyes that belonged to Pikachu, Red's Pikachu. As Blue stared, his light eyes narrowed with confusion, the small creature screamed, electrocuting everything around him. Blue snapped up one arm, covering his eyes from the incinerating flash, but when it died down, Pikachu was standing over a body, and one he recognised.
Brown eyes snapped open in the dead of night, sweat on his brow as Blue failed to comprehend what he had just seen. Pikachu... had killed Red? Why would he ever attack his trainer, that made no sense. But as he looked up, seeing the silhouette of the other nearby, the questions flared up in his mind all over again. Where were they? Where were his Pokemon? Blue slowly sat up, pushing the opening of his sleeping bag down as he made the decision without much thought of tact in his exhausted mind. "Red." He croaked the name out, but without waiting, he repeated the same question from earlier: "Where are your Pokemon, Red." His tone left the question a statement.
"What happened to them."
by Red on Tue May 19, 2015 8:29 am
In the daze between sleep and reality, Red attempted one last try at attaining at least some decent rest before committing himself to waking for the day. But that attempt was all for naught as he heard Blue's sleeping bag shift and suddenly, his paranoid senses slapped him clear awake. He rubbed the sleep from his eyes just as he heard Blue speak his name, even as the cries of his dying pokemon still clawed at his memory. "Red."
Red paused at the strangely serious tone Blue's single word carried, laden with something heavy. He glanced at the spiky silhouette he could make out in the extremely dim light, eyes narrowing in suspicion. "...Blue?" "Where are your Pokemon, Red." The soldier's throat clenched, his eyes widened and his chest felt tight, all because of the nosy bastard's innocent enough statement.
A cold hand of icy distance placed itself among his now rampant emotions-- anger, hate, anguish-- and put Red into an eerily calm state. If Blue really wanted to know, fine. He'd know. "I shot them," was his simple answer, the coldness he felt in his soul carrying into his voice. There was no regret or sadness there in those words. Just facts. He let the words hang in the air as he stared at Blue, his dark eyes just able to make out facial features. He watched, he waited for Blue to respond.
by Blue on Mon Jun 01, 2015 2:28 pm
Blue pulled himself further out of the old sleeping bag, propping himself up on his arms while Red turned to stare at him. He could not make out the other in the darkness, the light sifting into where they had camped turning Red into nothing but a dark shadow, contrast to the small amount of light. The fact that the other was still up should not have surprised him, but it confused the trainer. Umbreon and Arcanine were still keeping watch-- there was no need for Red to stay up. But as his eyes flickered around them, a pretty standard reaction to scan when first coming to his senses, the brunet's gaze landed on a small mound of fur near the foot of where he slept. Umbreon had no Pokeball, he could not rest like the others during the day, so he slept with his trainer when possible. Glancing at his Pokeballs, he could only assume that Pidgeot had gone out with Arcanine to replace the vulpine, since Exeggutor could not stand the cold.
But all of this passed through his mind in a mere few seconds, even as Blue knew that Umbreon would have woken with his voice. But the Eeveelution did not move, his rings lacking any light, as Red merely stated, "I shot them." That was not what he had expected, to say the least. Blue's tan eyes widened only a hair, his face losing colour in horror. He had been careful, but even he had only lost two of his team to undead-- and only one turned. How had Red lost all of them and then put them down himself? Even Pikachu? Imagining the other trainer's ace as one of the monsters made Blue's entire torso tighten. That would have been disastrous if he ever got away. Nothing would ever have stopped that Pikachu.
Except a bullet in his head.
Misunderstanding instantly, Blue's eyes half narrowed, almost with sympathy. He could not even imagine losing everything... losing them all. "... How long ago did they turn?" was his only response, a soft question as Blue could not look the other in his shadowed face. Maybe Red had just.. not known, stayed up on the mountain and they became infected by other undead in the area, or even the water source. A terrifying thought that only Red himself was capable of beating his own team.
by Red on Mon Jun 01, 2015 3:58 pm
"... How long ago did they turn?"
Red laughed.
It wasn't just the fact that Blue seemed to have grossly misunderstood Red's answer, no. While hilarious in and of itself, it was also the gentle, sympathetic tone Blue used to reply. Like he understood. Like he pitied Red. And that was fucking hilarious. The soldier's barked laughter didn't last more than a couple of seconds, but it saturated the air with anger and disdain. Red shifted, getting to his feet before taking a couple of steps toward Blue. In the dim light, his red eyes glinted dangerously beneath his worn and beaten hat, his expression cold and terrifying.
"They didn't," Red growled quietly, his voice little more than a whisper. Their dying cries clawed at him again, even as he stood sturdy in the waking world. "I was told to shoot them and I did. Better they die sooner than later."
The trainer half started when Red stood up, not only without warning, but also laughed. Blue was stunned, unable to move at first. His expression pinched, growing into a mix between confusion and suspicion. Was Red mocking him? "What the hell, man?" he growled quietly, the brunet's eyes narrowing to slits. But as Red continued his approach, Blue felt the urge to meet his challenge, and pulled himself out of the remainder of the sleeping back and to his feet just as the other spoke. Despite the near lunacy in the other boy's eyes, nothing prepared him for the realisation that followed Red's words.
"They didn't."
Blue instinctively reeled in confusion, half barking out, "What?"
Red's voice was quiet, but the coldness of it was no contrast to their freezing surroundings. "I was told to shoot them and I did. Better they die sooner than later." He refused to break eye contact the entire time, catching Blue's reaction with nothing to hide behind. The taller boy's eyes went round, shocked, as the words slowly processed.
"You'd never do that," he snarled, clenching his fists. The rumors, the stories he had heard before all of humanity vanished under a rock, all of them flashed in his mind again. They couldn't actually be true. Red would never do that. He loved Pokemon. It was the only sure thing in the world. But as Blue watched, the horror that seized his throat gave way to a bubbling rage, spawning from betrayal.
"You did." The words this time were hard, not desperate, but his tan eyes flashed with anger. "How can you even say something like that?! You're LYING to yourself! I don't fucking believe you!" he spat, but despite all of his efforts, the vision was growing all too clear. With how Red had become-- even back then he had a sense of humor, a heart, a soul. But Blue saw none of that now.
"You're a monster." That was the only explanation. "You're a fucking soulless monster. Out of all the people, I would have expected YOU to know better than to give in." Blue side stepped as he spoke, standing directly in front of Umbreon as his paranoia of Red shooting his partner grew.
"You'd never do that," Blue snarled after getting to his own feet, his own glare fixated upon Red. The soldier found it somewhat endearing that Blue would so readily defend Red from his own admittance, like it would actually do something. It almost made Red pity his old rival for thinking so decently of him. Almost. "Because you know me so well... right?" Red quietly replied, his foaming disdain and hate thinly veiled in his otherwise emotionless words. But soon, realization dawned on Blue's face in the dark, Red's fury and self-disgust flared in triumph. There it was. That was the look he'd been waiting for.
"You did." The two words were a sort of final statement, ending some other sort of argument Blue likely had had in his head. "How can you even say something like that?! You're LYING to yourself! I don't fucking believe you!" Blue's cries, his disbelief vindicated Red's own self-hate and disgust, proving to himself that he'd been correct about himself this whole time. That Blue's optimistic nonsense at the beginning of their horrible trek had been nothing but spewing, putrid trash. That he'd been right. Finally, Blue was seeing who -no, what- was truly standing before him:
A robot.
A--
"You're a monster." Red felt the words hit him, but it was like slicing a cadaver. The wound was there, but there was no longer any warm blood to seep out of it. No nerves to carry the pain. He already knew. And now Blue knew, too. "You're a fucking soulless monster. Out of all the people, I would have expected YOU to know better than to give in."
For all of Blue's angry spitting and snapping, Red could only feel light and relaxed. He was vindicated. No more doubt about whether or not there was any decency left. No more soul-searching, because obviously, there was none to search. The weight of possibility faded away like a ghost. Blue said so himself, after all. Red chuckled, giggled, even, but there was no mirth in the sound. It was hollow, "soulless" as Blue so expertly put it. "'Give in'?" Red mused as he watched Blue put himself between Umbreon and Red. Ha. Like his rifle would care about another body in the way.
"'Give in'?!" Red repeated, his voice teetering on lunacy. "You think that there was any choice? That there is NOW? But no, what am I saying, you're right, you're ALWAYS right, Blue-- I'm a soulless monster. They rounded everyone up and told us to shoot, and I did. I did. I shot them. I shot them down like fish in a fucking barrel because some asshole with more sparkle buttons than me said so." His voice cracked, his body shook as his mind went back and forth between reality, memories and the exaggerated nightmares. His eyes wide, he didn't even realize tears streamed from them freely-- not when his mind was caving in.
"They were just going to turn anyway," Red continued, not noticing if Blue spoke or not. He was only vaguely aware Blue was even still there. "That's what it does. No matter how careful you are, no matter how strong or fast or brave your pokemon is, in the end, it still gets them. They die and rise, no matter what. They all turn. It doesn't matter. It never matters. Better they died by my hand than something else." Shots fired. Pokemon fell. Espeon gurgled and choked on his own blood. Blastoise's eyes rolled back as his heavy form sunk to the ground, brain matter leaking out of the exit would in the back of his head. The look of betrayal on Charizard's face as a bullet pierced his gut.
The blood.
So much blood.
The choking stench of burning flesh as the flamethrowers incinerated the bodies.
Red's eyes suddenly focused, instantly aware of his surroundings once more as Blaine's laughter and his pokemon's screams clawed at his mind. He pulled out his Colt and pointed it at Umbreon's still form, not even bothering to look to his target as he stared wide-eyed at Blue. "What do you say, Blue?" he spoke quietly, his voice icy and void of emotion-- even anger. "Am I enough of a monster to shoot him? You'd know. You know everything, don't you? You always did, when we were kids." His eyes stayed on Blue expectantly as a cold realization dawned upon him.
Of course. It was all so simple. He wouldn't even need to ask Blue about it. How could he have missed it? "But you said I'm a monster..." So simple. "I was trained to kill monsters." Without hesitation, the Colt went from Umbreon to his own now open mouth, his hand working quickly and expertly to pull back the hammer, his eyes still blank.
Kill the monster.
Kill the monster, Red.
by Blue on Fri Jun 05, 2015 2:23 pm
Only adding to the horror in his gut, Blue was cornered, forced to only watch as Red... Red giggled? In another life, another time, he might have made a joke, but as it was the sound caused bile to attempt clawing it's way out of his throat. "'Give in'?" For once in his life, Blue not only had no idea what to expect, but no idea who this person was. Anger gave way to fear in his eyes, not even giving him the time to mentally prepare for the onslaught. "'Give in'?!"
"You think that there was any choice? That there is NOW?" The words were an arrow, stabbing deep inside of his mind, where Blue hid his deepest regrets. You could have ran, he thought numbly. "But no, what am I saying, you're right, you're ALWAYS right, Blue-- I'm a soulless monster. They rounded everyone up and told us to shoot, and I did. I did. I shot them. I shot them down like fish in a fucking barrel because some asshole with more sparkle buttons than me said so." Blue's entire stature was still, his hands no longer holding feeling. You could have ran like I did. Proven yourself a coward.
Red was crying. The stoic, steel strong Red was crying, and as he continued, something finally clicked in Blue's mind."They were just going to turn anyway." He didn't believe that. But his mind contradicted itself, immediately throwing But does he really? back at him. Blue's will was faltered, and it showed as his hands began to shake. This isn't stoic Red. This isn't strong Red. This isn't even insecure Red-... Blue's trembling increased only as the voice kept going."That's what it does. No matter how careful you are, no matter how strong or fast or brave your pokemon is, in the end, it still gets them. They die and rise, no matter what. They all turn. It doesn't matter. It never matters. Better they died by my hand than something else."
This isn't the Red I knew-- is he dead? Maybe he is. The trainer steeled himself, pulling his cold fingers together into fists, forcing himself to stop shaking. I won't be able to compare the two anymore.
But as calm as he was growing in the realisation, the icy stillness, it changed when the gun was whipped around, pointed right at him. Blue's eyes went round, his pupils shrinking a hair as they locked on the dark, steel barrel. But.. no. Where he was pointing, he would not harm him mortally, in fact... He'd miss me completely, or-- "What do you say, Blue?" The voice held no emotion, nothing that hinted at life, as Blue slowly followed the line with his eyes, watching the gentle rise and fall of his partner's flank for several seconds. His breathing grows rapid, speeding up as suddenly his heart is pounding in his ears. "Am I enough of a monster to shoot him? You'd know. You know everything, don't you? You always did, when we were kids."
How would he know? I'VE KNOWN NOTHING, ESPECIALLY SINCE WE WERE KIDS. Making the biggest effort of his life to not move, to remain calm, he growled, "You better be prepared to shoot me, too. If you harm him, I will never give up on getting revenge, killing you, until there is no more life in my body."
He never had a reply, though. Red suddenly seemed to change.
"But you said I'm a monster..."
Blue's gaze snapped up, catching the flicker of something in the other's crimson eyes.
"I was trained to kill monsters."
It was like staring into dull pools of blood when suddenly the gun was pulled from Umbreon.
Only to be turned on it's holder.
Blue gave a half shriek, bounding forward with his hands already reaching for the gun. The Umbreon had jumped to his feet behind him, no longer the target, but was only able to watch with perfectly circular eyes as Blue's fingers wrapped around the weapon, snapping it away. His brown eyes were owlish, haunted and panicked. But as expected, he hardened his features, yelling, "JESUS FUCK RED, don't kill yourself! Even you're not that stupid!" The insult was forced, causing his heart to pound louder in his ears. He begged for the retort. The insult. To see the fire in Red's eyes as he lashed out, pointed out Blue's own flaws-- anything.
by Red on Fri Jun 05, 2015 9:09 pm
It was going to end. He felt nothing, and Red supposed that was good. His own arm turned on him, or perhaps it knew what was best as it pointed his Colt's barrel down his throat. He didn't know. Nor did he care to fight it. He was still just following orders, after all. Kill the monster. Kill the monster.
But as his fingers were about to end it, a stronger hand wrestled it out of his limp grip easily. Red blinked as he realized Blue was actually still there, holding his gun and panting, his eyes wide as saucers. But the shock on Blue's face hardened to anger, and this time, it was Red whose walls couldn't stand. "JESUS FUCK RED, don't kill yourself! Even you're not that stupid!"
There was a silence in the air as the boys stood, staring at each other, and Red found that once again, words failed him. He stared helplessly at Blue, disarmed and alone and so utterly destroyed... It was all he could do to keep standing; he couldn't even try to regain his lost gun. As he stared at Blue, he felt the emotions push back against his mental dam, straining it. The agony, the despair, the guilt, the hate... It was becoming too much. His eyes teared again as the dam cracked and his breathing quickened slightly. No. He couldn't cry. Not in front of Blue. He couldn't let him see. He'd just laugh, like Blaine. He'd see Red go down and the talons and teeth would sink in, like Blaine had done. He couldn't take that again. Not again...
Please, I can't...
I'm so sorry...
Shaking, he closed his eyes against the tears as a soft sob cut him, and his legs gave out beneath him. His head hung as another sob gouged his lungs, and then another, and another... until finally, the dam was shattered. Ashamed and disgusted at his own lack of self-control, he gripped the craggy rock under his gloved hands in a vain attempt to regain some composure. It fell by the wayside as another racking sob punched him in the gut and soon the cave echoed with the sound of his wailing, horrible weeping.
They were gone. Why was that so hard to deal with, years later? He'd shot them, and he had to live with the consequences. Not Blaine, not Lance, not even Blue now let him escape his crimes. He was meant to suffer, to remember what he'd done... Their cries screamed louder, louder than even his own crying sobs. He couldn't be free. He could never be free.
by Blue on Fri Jun 05, 2015 10:49 pm
The silence stretched on longer that eternity, it truly felt like. Blue clutched the gun to his chest, the lack of words roaring in his ears like an ocean. Red did nothing, those dull eyes just stared at him. Far too long passed, Blue was barely aware of the concept of time at all, frozen in his horror as he watched the final snap in his best friend's mind.
Red's eyes suddenly overflowed again, the second time Blue had ever seen his rival cry and it happened both in the span of mere minutes. The other boy still did not speak, half slumping forward, the unnatural sound of horrible, grown up sobs tearing through his chest. Blue was at a loss, not realising how tightly he gripped the gun as he watched Red fall on his hands and knees before him. The old Red truly is dead, was his slow, numb thought.
But as Red made the attempt to get back up, only to fail, it suddenly became clear what he had to do. Working on instinct, the ugly cries still echoing in his head, Blue placed the gun beside his pile of supplies, gripping a familiar cloth from his pack before returning back to where the black haired man lay, broken.
Umbreon watched from the entrance of their shelter, his eyes too bright in the darkness. He vanished into the dark, keeping guard over the two boys. They were his boys, now. No one else would look after them, not Daisy, not Pikachu, not themselves. His trainer would not make things worse, not when the other was at his very lowest, the very bottom. Umbreon remained silent, an invisible ghost to protect them both.
Blue returned to Red, and only then did he hesitate. Reaching out, he placed on hand on the other's shoulder. It was a slow touch, and was quickly replaced by what he had retrieved from his bag. His spare jacket, the cloth clean as could be during the end of the world. Sitting beside where Red was, Blue remained silent, not looking at the other, but taking the sorrows he expelled into his own soul.
by Red on Fri Jun 05, 2015 11:14 pm
Red was lost in his own despair and agony, drowning into its depths even as he tried in vain to get the flood waters out from his soul. Tears streamed like rivers down his dirty face as the stabbing, clawing sobs tore at his thin frame mercilessly. It left him breathless and sick, but they still came, wave after wave of them. He couldn't see Blue anymore, not that it mattered. Red put the back of his hand to his open mouth to try and silence himself, but it too was a wasted effort. His sobs were screams in the blackened pit that he once called home, their echoes soaking into the very walls.
Red felt a hand on his shoulder, and didn't care who it was. It didn't matter that it was Blue, that he could use this to his advantage later. It'd been so long since he'd had contact from another human being, from, hell, his own pokemon, that he welcomed the tentative grasp as his own pathetic weeping attacked him still. He turned into the familial embrace, ducking his head down in a last-ditch attempt to hide his shame, and butted the top of his head against Blue's shoulder.
"I killed them..." he whimpered pathetically between his sobs, grabbing the jacket around him tighter. "I killed them... I killed them... Oh God, I killed them and..." He couldn't get it all out, the words becoming like knives in his very mouth. "They're dead... They watched me kill them..." The horrible screams of his sadness continued, growing to a macabre crescendo. "I killed my family! I murdered them in c-cold blood! And I... I can't... I can't ever..."
It was too much again. Red was rendered completely unable to form coherent words, much less sentences. It was all he could do to sit there, lightly leaning on his old rival for support.
Red did not flinch away from his touch, something that the boy was admittedly surprised by. As he crouched down, in fact, his rival took the wordless offer and half collapsed onto Blue without any warning. The brunet softened, reaching around with his arms to provide a safe embrace for the other to cry in. Red refused to show his face, and the loud, demanding sobs did not sway in the slightest, but between the crying, Blue heard him speak. "I killed them..."
The former champion flinched, wincing, but did not pull away. Red continued, gripping the jacket as if it were a lifeline. "I killed them... I killed them... Oh God, I killed them and..." Regret was forming in Blue's chest, tighter than a stone. Red was no monster, he was far from soulless, and as he continued crying, the trainer wished he could have bitten his tongue. Shut up for once in his life.
"They're dead... They watched me kill them..." Red was shaking, crying, a mess, breaking down. Fallen so far that he was even reaching for Blue himself, of all people, for help. The trauma of losing his family like that, of having to put them down... Didn't he say he was forced to?... Blue did not like what that implied, not at all. The familiar freezing chill of a deeply rooted fear crawled up his spine like ice water, but he remained silent.
Questions could wait. Answers could wait.
"I killed my family! I murdered them in c-cold blood! And I... I can't... I can't ever..." Blue's gaze grew dull, sad. He opened his mouth to speak, but all that came out was a croak. As if he had been the one crying, not Red.
"...you're not soulless... I'm sorry, Red."
by Red on Mon Jun 08, 2015 10:34 pm
Red didn't know how long he cried, but it felt like years. His throat was hoarse, his eyes burned and he couldn't breathe out of his nose anymore from all the snot. Eventually, though, the weeping died down, much to Red's relief and he merely panted against Blue's now sopping wet torso. He grimaced at the thought, both that he'd managed to completely disgrace himself like that and that it'd been Blue of all people to witness and trudge through it. How humiliating...
He swallowed against the dryness of his throat as he continued to pant slightly, wiping off all the fluids off his face. Tears, snot, drool-- crying had always been disgusting and messy. But Blue's comment did little else than remind Red of how far his dignity had fallen, and he withdrew from the embrace his rival awkwardly held him in. He shed the jacket, clean somehow, and handed it back to Blue. Now that his fit was through, there was little else than a void of exhaustion where all those emotions had just been. "Don't be," Red muttered as he sniffled the remnant mucus back. "It's... What's done is done." He stared almost blankly at the mouth of the small cavern they were in, seeing little else but darkness from the rest of the mountain's innards. "They're dead. I killed them. I just need to get over that." His voice was soft, but emotionless; he didn't have the strength for emotion anymore. Not after crying so hard. He gave Blue a sideward glance, not quite meeting the other's gaze.
"...Sorry about your shirt."
Slowly Red recovered, Blue trying to give him the time he needed, but at the same time the trainer was feeling his awkwardness from the physical contact flaring. He wasn't even sure if he had ever hugged Red before, the memory escaped him if he had. But slowly the other boy pulled back, allowed the brunet's arms to fall, lulling as they were released from the hold. Though it had not been unpleasant, Blue silently hoped that this never came back up in conversation later. But considering the way Red handed his cleaner jacket back, not meeting his eyes, he was selfishly grateful.
"Don't be," the other finally spoke, his voice devoid of the raging emotions that had overcome him before. "It's... What's done is done." While that was one way to see it... Blue now knew the full grand scale of the situation. Red stared out of the shelter, his own eyes burning into the back of the other's head. Would he be able to hold it together now..? The gun Blue had taken was still over by his pack, well out of reach of a mere grab, but still. The image of the colt's barrel in his rival's mouth haunted him. "They're dead. I killed them. I just need to get over that."
Blue gave a soft sigh, pulling his own shoulders back to roll them, releasing the tension from the position he had taken for the time while Red had sobbed. Slowly standing to his feet, gripping the garment in one hand as the other gave me a slow glance. "...Sorry about your shirt."
That caught him off guard, the tiredness of Blue's expression shedding away as his face went slack from the unexpected comment. "Wha-? Oh." Looking down at his partially soiled shirt, the trainer gave a small cringe, but shrugged it off. "Whatever. Been through worse. I'll wash it next we find a stream." Turning away from Red, the trainer approached his things once more, fulling intending to change into his spare shirt until he could 'clean' the one he wore then. Or at least as clean as things could get.
by Red on Sat Jun 13, 2015 2:54 pm
"Wha-? Oh." Blue seemed relatively unaware of the darkened wet spot on his own shirt, which Red was at least somewhat grateful. Blue wasn't being nearly as vicious as Red thought he'd be; maybe he'd just spent too long of a time around Blaine. Maybe the scientist poisoned his mind? Make him more complacent? It was a thought, but Red was too tired to continue thinking on it. "Whatever. Been through worse. I'll wash it next we find a stream."
Red only lightly nodded before dragging himself to his feet, eyes and face now fairly dried; the only remnants of his fit were his red, puffy eyes. But even those were hard to spot in the dark. Without much of a thought, Red went to where Blue had placed his gun and picked it up, staring at it a small bit before sticking it into its holster. While he was aware he'd nearly committed suicide with it, leaving such a useful took behind was as much suicide as sticking the thing in his mouth. Wearily, wordlessly, Red went back to his spot against the wall, almost falling against the cave and sliding down before becoming still. He was so exhausted, and he was grateful for it. He was too tired for anger or anguish, or really even embarrassment. Maybe he could even get some sleep...
With a sigh, the soldier felt his head drooping as sleep began to claim him. He didn't resist. Soon, his breathing evened and for once, he was glad to not be completely alone that night.
[[Time skip next round.]]
Blue reached into his pack, stifling a sigh. Watching Red crumble had been the last thing he had expected when finally running into the other. His thoughts tool over, leaving him frozen, still as his bag was open before him, the trainer's eyes glazed over. Pikachu. The little guy had been the bane of his battling career. Now he was just... gone? It would have been a dream come true years ago. How many times had he wished death upon the powerful electric type? Blue slammed his eyes shut, not prepared for the guilt and grief that clawed through his chest. It's not your fault. But he still felt somehow responsible.
Snapped from hi thoughts, Blue jumped, Snapping his head around to look over his shoulder. Red picked up the gun, the one that the trainer had snapped out of his mouth, and for a second panic and fear shot through the boy. But as he watched, Red merely stared at it a second and stuck it into it's proper case. Blue did not stop watching until the other slumped down, turning back to his case at hand only to see he was shaking. If Red tried again in the middle of the night, would he be able to stop him? Would Umbreon know to? Exhausted, Blue quickly swapped out his soiled shirt for a new one, eventually closing his back while a sigh escaped him.
This was not what he had wished for. Blue slipped back into his sleeping back without a word, pulling the cover over his head. He did not fall asleep immediately, instead memories of Red and Pikachu dancing through his thoughts. Those small, intelligent eyes, always knowing more than Blue himself.
by Red on Sat Jun 20, 2015 10:54 am
Mount Silver [Base] | Morning (47)
Light streaming into his eyes was always Red's prime choice on how to be woken up. Scrunching his face a little as the sun greeted him cheerily, Red lifted his head to see that Blastoise's hole revealed the bright light of a new day. Great. Without much else of another thought, Red's body rose out of pure habit and routine, collecting his things swiftly and silently while his mind still struggled to rouse properly.
It didn't take long to gather his things, since he hadn't truly unpacked. He hadn't even actually slept in his sleeping bag-- merely on top of it. But as he turned to leave, he noticed the other bag and the body within it and suddenly remembered that yesterday had actually happened. Red's face flushed with embarrassment as his breakdown flashed back in his mind. The tears, the relentless sobs, the... the gun. A hand went to Red's now throbbing temple as the humiliation only grew. Gross. He'd just practically poured his fucking heart out to Blue of all people and now he was stuck with him. Fantastic. Way to make things easier on yourself you miserable twat, Red berated himself inwardly taking a couple of steps toward the sleeping Blue.
He gave Blue's foot -or what he assumed would be Blue's foot- a firm but not malicious kick, not patient enough for the boy to wake on his own. With so much embarrassment dogging his heels, Red was eager to begin walking away from it. And as appealing as the thought of leaving Blue there was, Red had a nagging feeling that Umbreon was still lurking about and Red really wasn't up for another lecture from Blue. And this time, it'd likely be laced with pity and sympathy and other vomit-inducing things. "Get up. We need to move." His voice lacked any real emotion still, an after-effect from last night.
by Blue on Wed Jun 24, 2015 10:01 pm
Mount Silver (Base)|| Morning
Coming to with a start, the boy's tan eyes snapped open from the smack on his foot. Unaccustomed to the style of awakening, Blue shot up, his trusty crowbar already in hand, pulled back and poised to spring. But there was a person in front of him. What the--?!
The memories of the previous day were slow to return, but in only a few seconds did it all replay and register in his mind. Red told him to get up as his body already slumped, the weapon hitting the hard ground with a metallic clang.
"...Right." Already exhausted, he pulled himself from the sleeping bag with an internal sigh. Traveling with Red. That would be one hell of an adventure.
It did not take long to scoop his few things together, having only really pulled out the old sleeping bag, and Blue pulled the pack onto his back soon enough. Feeling the reliable, clean steel in one hand, his fist was tight around the weapon. He was on edge already.
Walking towards the mouth of their shelter, Arcanine trotted up. He nodded, tiredness from the sleepless night showing in his eyes. Blue ran a hand through the dog's fur without thinking, murmuring, "Return," as he recalled the fire type.
Umbreon also showed up, but his lithe body showed no sign of exhaustion, his legs steady and his red eyes calm. "Any trouble?" The Umbreon flicked his tail. "Nothing that was not handled quietly." Blue nodded, turning to follow Red without much thought about why they were heading in any direction in particular.
by Red on Wed Jun 24, 2015 11:31 pm
Red was honestly surprised Blue had decent reactions-- he half expected Blue to just snort and roll over. But his rival started quickly at the kick to his foot, crowbar in hand as he slowly came to reality. Satisfied that Blue was awake, Red took a few steps toward the mouth of the alcove to lean on the cave wall and watch. The only sounds around him were Blue's shuffling and the heavy footfalls of Arcanine and Umbreon coming close. He eyed them silently a moment before looking further beyond them. It was strange to listen to how quiet his mountain had become... Strange and haunting.
"Return," came Blue's soft command to one of them; Red could only guess by Arcanine's fatigued stride that it was him. He didn't turn to look at the exchange, however, prefering to keep his attention on their surroundings.
"Any trouble?" Blue asked, and Umbreon replied just as softly, "Nothing that was not handled quietly." Red took that as a cue to lead the way, pushing gently off the cave wall with his rifle in both hands, ready to fire. Wordlessly he stepped off, setting a brisk pace through his mountain.
It was a few hours of the grueling pace as he took twisting and turning caverns and climbed short cave faces before reaching the first of the checkpoints up the mountain: a small covered outlet that dropped about three stories down to the forest below. He'd kept a healthy lead, making sure Blue or umbreon never got close enough to begin any sort of conversation. Red wanted all memory of last night and his weakness to rot and die in that fucking alcove. He was panting lightly, thankfully used to the weight of his armor and athletic enough to be able to continue for a small while longer. Taking his water pouch and swallowing a gulp, the soldier turned to Blue, unsure whether or not he needed to offer his water. "Do you need to rest?"
by Blue on Tue Jul 07, 2015 7:03 pm
While following Red, the trainer made no effort to be closer to the other than necessary. Long as Red was in his line of sight, Blue didn't care. The novelty of finding his rival wore off with sleep, but even when his eyes burned into the back of the other's head, Blue ran the words from last night through his head.
With Arcanine resting for the day, Umbreon trotted beside his partner. The vulpine's steps were precise and silent, his small paws making no sound even as his rings gave a natural, dull shine. Repositioning his pack, Blue reached for his other oldest Pokeball. "Pidgeot."
The bird emerged before him, his eyes blinking open before falling onto the man and softening. Flapping his large wings slowly but with power. "Morning," he murmured gently, nodding respectfully to Umbreon as well, who echoed the action.
"Your day to keep watch," Blue explained, never slowing his pace, though willing to tear his eyes away from Red's form to look his Pokemon in the eye. The hardness to his features lessened and he smiled at the bird. Reaching out with one hand he gently petted the soft feathers on the Pidegeot's head. The bird cooed in response, only taking to the air above the group once Blue pulled his hand back.
Upon turning, the trainer caught the dark type's unreadable crimson eyes on him. Suddenly his skin itched. "What?" He demanded, though he continued keeping his voice low. Umbreon did not reply, flicking one long ear as he slowly turned his gaze away and trotted forward so he was a few steps ahead. Blue sighed. "How much did you hear of last night?"
"I don't sleep, Blue," was his quiet reply. Though the statement was not entirely true, the human did not question it and sighed.
"I'm a fucking idiot. If that had happened to you, and he asked that, I would have shot me, too," he muttered, running one stressed hand through his hair.
"It would have come up eventually," his partner pointed out, still not turning to look at him. "Better there, where you were relatively out of harm's way, than out here in plain sight."
Blue half grumbled, not saying any words in particular, but he did not argue. Umbreon tended to be more clear minded than him anyway.
As the rest of the time drew out, Pidgeot did his job, and no real trouble fell upon the small group for the time yet. Eventually Red stopped, catching Blue's interest, and he approached at the same speed he had kept up all morning. The trainer caught up as Red was taking a swig of water, which as Blue considered was not a bad idea. "Do you need to rest?" he asked, earning a pause from the other.
"I can keep going," he said after a moment, but crouched down. Umbreon tilted his head to one side, watching as his trainer pulled out a water bottle and a bowl. Placing the bowl in front of the vulpine, he poured some water inside of it before bringing his own water to his lips. Umbreon nodded his thanks before dipping his head to take the refreshment.
by Red on Wed Jul 08, 2015 8:34 pm
"I can keep going," Blue replied as he supplied water into a collapsible bowl for Umbreon, to which Red silently nodded and took another swig of his own water. He turned to stare outward, out of the alcove and to the ruined forest that sprawled like a corpse below. The treetops were already beneath them, bathed in the eager morning light. For once, the sky was relatively clear with the exceptions of fish scale clouds dotting the endless blue above them due east. Red stared the clouds down, his mind working. Weather atop the mountain was harsh, unpredictable and unforgiving. Though it was decent now, it could change in a matter of hours even all the way down here.
Red pocketed his water, watching the world below mostly because he wanted to avoid as much eye contact with Blue as possible. As he heard Umbreon taking his drink, he adjusted his own armor and belongings. "We'll be out of the mountain for a small while to reach the next tunnel. It's an easy path. Next checkpoint's in another hour or so, barring emergencies." He kept his voice low and controlled-- emotionless. It was easier since most emotion had bled out of him the night prior. "I'd suggest keeping Pidgeot in the ball. Don't attract attention."
by Blue on Sun Jul 12, 2015 12:55 pm
While Umbreon got his fill, Blue's eyes flickered up. They burned into the back of Red's hat covered head, as if he could decipher what was going on inside, before following his line of sight. Were they going out there? The other turned, pocketing his own water and making Blue drop the possibility of eye contact. The last thing he wanted was to discuss what happened last night. I still can't believe Pikachu is....
"We'll be out of the mountain for a small while to reach the next tunnel. It's an easy path," Red explained then, earning a nod from the trainer. He crouched down, gently patting Umbreon's head before picking up the bowl as it was completely emptied. "Next checkpoint's in another hour or so, barring emergencies."
"Sounds like fun," Blue muttered sarcastically, unable to hold back a smirk and giving a soft snicker. Travelling with company could be enjoyable, right? Least Red of all people would be fun to mess with. Maybe. Right?
Flicking his wrist, Blue scattered the last remnants of moisture before sticking the collapsable bowl back into his bag. Umbreon straightened up, his short whiskers twitch as his eyes burned into his trainers. Don't do something stupid, his eyes warned. "I'd suggest keeping Pidgeot in the ball. Don't attract attention," Red added, earning an agreeing incline of the vulpine's head.
"Fair enough," Blue murmured. Gripping one of the balls at his side, he gestured with the other for the large bird to come down. As Pidgeot approached, Blue explained softly, "Take a rest for now. I'll let you know when it's safe again." He nodded and with a brief flash retired back to the classic Pokeball. Blue stood back up, Umbreon's eyes still searing into the back of his head. "Lead the way."
by Red on Mon Jul 13, 2015 10:06 am
"Fair enough," Red heard Blue reply, to which Red couldn't help but allow a small sigh of relief escape him; Blue wasn't putting up a fight. Thank God. It was nicer when his rival just did things when asked and didn't question or argue it. There was a flutter of wings in seconds, indicating Pidgeot's descent. "Take a rest for now. I'll let you know when it's safe again." With the sound of the bird being returned into his ball, Red turned his head to finally look back at Blue, who was the object of Umbreon's stern glare. An eyebrow perked at the sight, but Red didn't question it aloud, instead giving Blue time to comment, "Lead the way."
Wordlessly, Red nodded, turning back to the path to do just that. He kept his rifle close and ready to aim as he quickly made his way through the brush and trees. Though they were smaller, it was still easy to see they were lower on the mountain by their sheer number. The footing was still easy and forgiving as well, even lending some shade in the morning sun. Red passed through like a ghost, knowing where to step and how fast to pace himself. He made some pauses along the way, if only to watch the next bit of route before continuing.
The silence hung in the air once more and it put Red on edge. They should have run into something by now, but there was still just... nothing. Where were all the pokemon? Surely all of the Infected didn't just vacate? The inexplicable absence of wandering Undead made his heart pound and his skin crawl. It wasn't right. There was something wrong with his mountain, and he wasn't sure if they'd live to tell the tale... His eyes went up to the clouded peak, determined. No. There was something he needed to do here, and he needed to reach the top. If his life had ever been worth anything, it would be to reach that top.
One last time.
Red's gaze returned to the path ahead, still searching for movement that wasn't there. As they stood still, Red looked to Blue and Umbreon, his eyes settling on the pokemon. "Just how many Undead or Infected have you spotted so far?" he asked in a low voice, just barely audible. He needed to know-- Umbreon's earlier report had been frustratingly vague.
by Blue on Sun Jul 26, 2015 6:58 pm
Red led the way through the terrain, the show off. Blue felt himself frowning, half scowling from behind as he kept behind. The soldier crouched, paused, listened, God it was just like before. His mountain this, his mountain that. Tan eyes narrowed until a sudden jab in his side made him suck in a sharp breath.
Snapping his head around, he saw Umbreon glaring back him, meeting the defiant look of the trainer. "You knock that off," Umbreon hissed, keeping his voice down from where he crouched nearby. Blue did not bother playing dumb, though part of him wanted to. "I know what you're thinking." Umbreon always seemed to read his mind.
Despite that, though, his shoulders slumped forward. Why did everyone always take Red's side? Maybe because you're being a goddamn baby. Not like it was wrong. Suppressing a sigh, Blue half started when Red turned back, staring right at him.
But then without hesitation, it landed on Umbreon. "Just how many Undead or Infected have you spotted so far?" Blue tensed, the memory still raw with the image of his gun pointed at the vulpine.
But the dark type showed no fear, as unreadable as ever as he trotted forward. "Near nothing as far as we ran into," he confessed, matching Red's low tone as his crimson eyes flickered above them. "A bug or two, but nothing in far too long. The quiet isn't right."
Blue suddenly realised he had been missing something the entire time. Too quiet? He shot a look over his shoulder, paranoid. "What could cause that?" he asked quietly, eyes burning into Red for answers.
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The vest contains a total of twelve pockets; eight front outer, two rear outer and two front inner, for storage of survival, signal and communication components. All pockets have fastener tape closures. For rescue-lift, a nylon webbing harness system is permanently attached to the vest in a cradle fashion and contains both leg and chest straps. The chest strap also provides the means for attaching the LPU-10/P life preservers. The vest also provides means for attaching a motor housing and face mask carrier when wearing MOPP clothing. A pocket is also provided on the vest for carrying a AN/PRC-90 or AN/PRC-112 radio.
The knitted, aramid cloth used for the body of the vest shall be Olive Green 106 and shall conform to MIL-C-43989. The oxford aramid cloth used for all pockets shall be Olive Green 106 and shall conform to MIL-C-43842.


text:
Home » Biotechnology
Vectors for gene cloning- Plasmids and Bacteriophage
May 4, 2022 March 27, 2022 by Somak Banerjee
In molecular biology, a vector refers to a DNA molecule that transfers genetic material into another cell and the genetic material gets to incorporate in the cell genome and expressed. The foreign DNA carried by the vector is called recombinant DNA. The vectors are generally characterized by three factors such as the origin of replication, multi cloning site, and a selective marker. Plasmids and bacteriophage are commonly used molecular vectors which are described as following.
Factors important for selecting a vector
Size and copy number
Conjugation and Compatibility
Types of Plasmids
Lytic and Lysogenic Phages
Genetic organization of lambda DNA
Linear and circular forms of lambda DNA
M-13: Filamentous phage
Plasmids are circular DNA molecule which are present independently inside the bacterial cell and has one or more genes. These genes often play an important role in the host bacterium. To illustrate, bacterial plasmids mostly contain antibiotic-resistant genes which protects the bacteria from antibiotics like ampicillin. In genetic engineering antibiotic-resistant gene functions as a selective marker, to find out whether the plasmid is transferred in the bacterial cell. Apart from that, most of the plasmids contain a specific DNA sequence called the origin of replication which helps plasmids to replicate independently. Some plasmids often used host cells enzyme for replication while some can code for their own special type of enzymes for replication.
The size of the plasmids ranges from 1kb to 250 kb. But the size of the plasmid is important for cloning purposes. All types of plasmids are not useful as a cloning vector; plasmids with a size of fewer than 10 kb can be used as vectors. However, in some cases, larger plasmids can also involve as vectors. Copy number refers to the number of plasmid molecules present in a particular bacterial cell. Larger plasmids are generally considered as stringent and often present in low numbers while small plasmids are generally present in high numbers. The high number of plasmids helps to incorporate several recombinant DNA into a cell.
Conjugation is a process by which bacterial plasmid can be transferred from one cell to another cell. Conjugative plasmids involve in the process of sexual conjugation. On the other hand, non-conjugative plasmids are also present in bacterial cells. The non-conjugative plasmids lack a type of gene called tra gene which is involved in the transfer of plasmids by conjugation and present in conjugative plasmids. A bacterial cell can have several types of plasmids among which different types of conjugative plasmids are also present. In this case, the transfer process required the compatibility of the plasmids present in a bacterial cell. Two incompatible plasmids will not be present inside a cell; one or the other will be lost. Hence, the compatibility of the plasmids plays an important role in the selection of cloning vectors.
The naturally occurring plasmids are classified under five major groups on the basis of the genes present in the plasmids. The five groups are the following:
F plasmid or Fertility: F plasmid contains specifically the tra gene and facilitates the transfer of plasmids by conjugation.
R plasmid or Resistance: This type of plasmids contains one of the multiple antibiotic resistance genes. In clinical microbiology, R plasmid plays an important role as it is the main reason behind the development of antibiotic resistance in bacterial pathogens. An example of R plasmid is RP4 which is commonly present in Pseudomonas sp.
Col Plasmid: Col plasmids are responsible for the production of colicin and commonly found in some strains of E. coli. This is a type of toxin which is generally released in the environment to reduce competition for nutrition. ColE1 is an example of this type of plasmid which is found in E. coli.
Degradative plasmid: This type of plasmid is responsible for the degradation of complex compounds like toluene, salicylic acid. Example: TOL found in Pseudomonas putida.
Virulence plasmid: Virulence plasmids are present in pathogenic bacteria and responsible for causing infection or disease to the host cell. Ti plasmid is an example of virulence plasmid which is present in Agrobacterium tumefaciens and causes crown gall disease in dicotyledonous plants.
Bacteriophages are a group of viruses that used bacterial cells as host and reproduce by infecting bacterial cells. Like all other viruses bacteriophages are also consisting of a protein coat that provides protection to the genome. Most of the phages are DNA viruses that code for several genes responsible for virus replication. A major problem of using plasmids as a cloning vector is the size of the gene of interest is small. However, bacteriophages help to overcome this problem, as larger genes can be transferred by using bacteriophage as a cloning vector.
The pattern of infection of different bacteriophages is mostly needed three steps to complete. Firstly, the attachment of the phage at a specific site of bacterial takes place. The phage is then injected into the DNA inside the bacterial cell. In the second step, the genes present in the DNA of the phage codes several enzymes which helps to replicate the phage DNA. Finally, other genes also code several proteins by which the capsid is formed and the new phage particle is assembled inside the capsid. This step is followed by lysis of the cell and release of the phages from the cell. In several bacteriophages, this cycle occurs rapidly and it is called a lytic cycle. On the other hand, in some phages, the phage DNA got incorporated in the host genome after entering the host cell and remains there for several cell divisions. The phage DNA which has incorporated in the bacterial DNA is called prophage and it is quiescent. The bacteria which carry the phage DNA remain as same as any other uninfected cells. This type of infection is called a lysogenic cycle. The phage DNA finally released from the host cell by entering into the lytic cycle. The infection cycle of lambda phage is an excellent example of a lysogenic cycle. In the case of cloning, the lysogenic cycle is most important, because, the phage DNA can carry a specific gene that is further incorporated in the host genome. As a result, the host genome will code a specific gene carries by the phage. Therefore, lambda phages are mostly used as a cloning vector.
The lambda phage is a typical example of bacteriophage which has a polyhedral head and tail. The DNA is protected by the polyhedral structure, and the tail interacts with the specific site of bacteria for attachment. Lambda phage usually infects E. coli. The DNA of lambda phage is 49 kb of size. Gene mapping and DNA sequencing analysis provide all the information regarding the genetic arrangement of the phage DNA. According to the data gathered from these analyses, the genes of similar functions are clustered together in the genome. To illustrate, the genes responsible for the formation of the capsid are clustered together at the left-hand side of the polyhedral head while the genes responsible for the incorporation of the phage DNA with the host genome clustered together at the middle.
The formation of the lambda DNA plays an important role as a cloning vector. The genome of a lambda phage is made up of two complementary linear DNA. As the DNA strands are complementary they attach together and forms circular double-stranded DNA. In molecular biology, the term sticky ends refer to the complementary single strands as they attach together. In lambda DNA the sticky end is termed as cos site which plays essential roles in the infection cycle. Firstly, the phage DNA remains circular inside of a bacterial cell after insertion due to the cos sites. Then, the second role of the cos sites is rather different and comes into play after the prophage has excised from the host genome. At this stage, a large number of new lambda DNA molecules are produced by the rolling circle mechanism of replication, in which a continuous DNA strand is "rolled off" the template molecule. The result is a catenane consisting of a series of linear lambda genomes joined together at the cos sites. The role of the cos sites is now to act as recognition sequences for an endonuclease that cleaves the catenane at the cos sites, producing individual lambda genomes. This endonuclease, which is the product of gene A on the lambda DNA molecule, creates the single-stranded sticky ends and also acts in conjunction with other proteins to package each lambda genome into a phage head structure. The cleavage and packaging processes recognize just the cos sites and the DNA sequences to either side of them, so changing the structure of the internal regions of the lambda genome, for example by inserting new genes, has no effect on these events so long as the overall length of the lambda genome is not altered too greatly.
M-13 is a type of bacteriophage and it is totally different from the lambda phages. The structure is filamentous, the genome size is very small (6407 nucleotides), single-stranded and remain in a circular formation. Due to the small size of DNA, the phage genome can carry only a few recombinant genes. The M-13 phage DNA enters inside an E. coli cell via pilus. Inside the cell, the phage DNA codes for specific enzymes that synthesize a complementary strand of DNA. This newly formed double-stranded DNA molecule then proliferates into several copies. During the bacterial cell division, each daughter cell contains copies of phage DNA and the phage DNA continues to replicate inside the daughter cells. Finally, new phage particles are assembled and released from the bacterial cell. M-13 phages are also used as a cloning vector. The genome size is 10 kb which is enough to insert new genes. Apart from that, the modification of single-strand DNA to double-strand DNA inside the host makes it unique.
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Top and Best Biology Websites or Blogs of 2023 for Study Notes
T.A. Brown, Gene cloning and DNA analysis: An Introduction, 6th Edition, Wiley-Blackwell.
Bolivar, F. and Backman, K., 1979. [16] Plasmids of Escherichia coli as cloning vectors. In Methods in enzymology (Vol. 68, pp. 245-267). Academic Press.
Meissner, P.S., Sisk, W.P. and Berman, M.L., 1987. Bacteriophage lambda cloning system for the construction of directional cDNA libraries. Proceedings of the National Academy of Sciences, 84(12), pp.4171-4175.
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Somak Banerjee
Somak Banerjee has completed his M.Sc. in applied microbiology from the Vellore Institute of Technology, Vellore, India.
1 thought on "Vectors for gene cloning- Plasmids and Bacteriophage"
Rajan giri
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