5.1 New LB Agar was prepared.
12.8 Comment 13.08.2010 (Hanna): We wondered how the whole Rep stuff could be sequenced reverse AND forward without any forward sequencing primer... By checking the "sequencing book" we found out that always the wrong primers were used: the GOI primers can be used in order to sequence the Gene of interest = GOI (!) in pAAV_MCS and NOT the pSB1C3!!!!!!!!!
Eventually GATC partially managed to sequence pGA14_Prefix-left ITR clone 1 and 2.
The sequencing results were better than before but in the ITR region still not evaluable due to the strong secondary structures.
It seemed that, despite of successful insertion of the RFC10-Prefix, something went wrong because there were 2 bases missing in the NotI restriction site. But by having a closer look, it became obvious that the Geneious misinterpreted the chromatogram and overlaid the peaks of the missing bases.
Therefore clone 4 was dicarded from the plasmid- and glycerol stock.
Comment: In order to verify the results, P147 and P150 were sent for sequencing. Because sequencing never worked before, they will be sequenced under special conditions this time.
Afer 50 minutes a picture (see below) was taken. Because there was no clear pattern (two bands) for the pSB1C3 backbone, the band of the insert mGMK was cut out and the gel ran another 20 minutes. After that, there were still two bands. Both were cut out and it was continued with two possible backbones. They were labeled vector (lower) and vector (upper).
Transformation Two approaches were prepared, one for each vector and put in 37 °C room overnight.
Comments: Sequenzing revealed no mutagenesis of SalI in pAAV_RC_1.1_SalI, so SDM now will be repeated.
Plasmid was transformed into BL21 cells. Clones have to be picked tomorrow.
Aim of the experiment: We wanted to produce biobricks from the expression plasmids Rep40ex, Rep52 ex, the Rep 68 and 78 in which the PstI at position 310 was silenced and the AAP. This was performed by a PCR with primes that annealed to the ends of the later biobrick but also contained restriction sites that can be cut afterwards and cloned into pSB1C3.
Five µl of PCR-product were used for an analytical gel to see if the PCR worked.
Comment: The result is that the PCR worked for the longer Rep proteins (68&78) with which a Quick-change was performed to remove the PstI(310) but not with the constructs for the smaller Rep variants (40&52) that were recieved from PD Kleinschmidt. As the longer Rep variants the AAP PCR Reaction also resulted in a PCR-product of the expected size. Unfortunately there were a secondary band of ~90bp for Rep_68_ex. For this reason we performed a praeparative gel and decided to cut the bands of all PCR products.
The bands marked in the gel picture were cut and a gel extraction was performed.
Comments: We sent for sequencing pSB1C3_RFC25_longlinker (P105) to check the strange result of the test digestion of pSB1C3_SDM_PvuII(01.08.10.
pSB1C3_SDM_PvuII is too long for sequencing wherefore we sent pSB1C3_RFC25_longlinker because the longlinker just have 54kb (bp, nicht kb (Volker) (necessary is just the backbone we don't have.
pGA14_lITR_RFC10 (P147), pGA14_rightITR (P150) and pSB1C3_RFC25_CFP (P51.1) were digested with EcoRI and PstI (Buffer 4) according to the standard protocol. Digestion Time: 80 minutes.
pSB1C3_RFC25_CFP: about 2100 bp and 800 bp.
pGA14_leftITR_RFC10: the size of the insert should be 135 bp.
pGA14_rightITR_RFC10: the size of the insert should be 138bp.
left ITR + pSB1C3: 5µl Buffer, 1 µl Quick-Ligase, 2,5 µl left ITR, 1,5 µl pSB1C3_RFC25.
right ITR + pSB1C3: 5µl Buffer, 1 µl Quick-Ligase, 2,5 µl right ITR, 1,5 µl pSB1C3_RFC25.
Transformation: performed according to the standard protocol (BL21). The cells were plated on a agar plate with chloramphenicol. The clones will be picked tomorrow.
Plasmids were transformed into BL21 cells. Clones have to be picked tomorrow.
The Motivation: investigation of the influence of different GOI amounts.
The Plan: keep rep/cap and pHelper amount stable (3,3 µg each => 6,6 µg), differ the GOI (YFP) amount in each stock.
This appproach also didn't work, same result. the guess is that BpmI doesn't work anymore. will be repeated by Chris W. on 05.08.10. the result will show that the guess is right and BpmI from labstock is changed out with a new one.
Test transformation was performed in order to test the efficiency of produced chemical competent XL1 blue cells.
Transformation was performed according to the standard protocol and the plates were incubated at 37 C.
8 sequencing files were analyzed "per hand" base by base. Alignments (will be inserted soon) delivered that the right ITR is 100% OK and was successfully converted into the RFC10 BioBrick standard.
The sequencing and alignments of the left ITR delivered that a 15 bp fragment in the middle of the sequence is lacking. Therefore further test digestions have to be performed. Nevertheless also this ITR was successfully converted into the RFC10 BioBrick standard. Secondary structure analysis showed that the stemloop-structure will be nevertheless forming. We will try to test whether the referring fragment is also missing in the pAAV_MCS. If it's also lacking there we will continue with this ITR and test whether it functions.
Comments: The PCR of Rep 40/52 didn't work (see 03.08), whereas the PCR of Rep 68/78 and AAP was succesful. Ligation was done with two different samples of pSB1C3 (see agarose gel). Samples from digestion and from ligation are stored in the 4°C freezer.
Comments: Digestion was done with XbaI and SpeI, it has to be checked if the inserts are cloned into the vector in the right orientation.
For the purification 95 µl of buffer PBI was used.
For the Ligation 10µl buffer (2x) and 1µl Quickligase were used.
The transformation was done following the standard protocol using B21 cells.
New LB Agar was prepared.
Results: Sequence read looks good. The incorporation of the PCR product can be confirmed. It can be seen that the insert is in the RFC10 standard and prefix and suffix have the right sequence. Therefore, this BioBrick can be send to the registry and used for BioBrick assembly.
Of each construct two clones were picked. plates are still stored in the cold-room, tomorrow mini-prep will be done.
Comments: PCR was performed one time without DMSO and one time with DMSO. Only the approach with DMSO showed bands in the analytic gel after PCR. That's why only this approach will be noted.
Ligation with T4-Ligase and transformation with BL 21 was made. Clones have to be picked tomorrow.
Against our expectations both agar plates contain very few colonies. In order to figure out if the lack of colonies is due to the XL1 blue cells or loss of pUC activity, test transformation will be repeated this evening with recently prepared XL1 blue cells as well as with Lab-XL1B cells. 50 pg pUC-plate contains 8 colonies 100 pg pUC-plate contains 18 colonies Transformation will be performed again with 100 pg pUC (= 2 ㎕ pUC).
For the tripple mutant of pAAV-RC a Quick-change reaction was carried out to remove the last restriction site (SalI) that is required for the Viral Brick standard. For this construct glycerol stocks and minipreps were carried out for two clones.
TO do: Test digestion and sequencing!!!
PCR of pKEX-2XL.Rep 40 (P22) and pKEX-2XL.Rep 52 (P23) following gelrun (1%) and gelextraction.
72°C 15 sec Repeat this cycle 8 times.
72°C 15 sec Repeat this cycle 17 times.
72°C 20 sec Repeat this cycle 8 times.
72°C 20 sec Repeat this cycle 17 times.
Digestion of pSB1C3 (P51.1) with EcoRI-HF and SpeI following gelrun (0.8%) and gelextraction.
All marked fragments were extracted. The Gelextraction was performed according to the standard protocol following a digestion of the PCR products with EcoRI HF and SpeI and a purification of the PCR product. The Ligation was not performed according to the standard protocol: 1 µl T4 DNA Ligase, 1 µl (10x) Buffer, 3 µl vector pSB1C3 (60 ng/µl) and 5 µl insert (all concentrations about 100 ng/µl). The Transformation (with BL21) was performed according to the standard protocol. Tomorrow there will hopefully be some clones on the agar plates.
Comments: BioBricks are ready to use. The sequenced pSB1C3_beta globin (P133) and pGA14_mVenus (P60) will be digested with different enzymes and will be assembled in order to produce the first step in assembling the whole vector together.
Results: As it can be seen in the picture below, digestion of the vector pSB1C3_betaglobin (left lane) revealed a band at around 2500 - 3000 bp. The expected size after digetsing with SpeI and PstI was: 2555 bp. The band ran a little bit higher than expected, anyhow the band was cut out of the gel. The right lane belongs to the pGA14_mVenus which was digested with XbaI and PstI which resulted in two fragments. mVENUS was digested. Expected sizes were: mVenus = 756bp and pGA14 = 2900bp. The expected sizes correspond to the bands seen in the picture below. The smaller fragment belonging to mVenus was cut out of the gel as well.
For ligation the T4-Ligase were used.
Additionally a control ligation was performed.
Ligation duration was 45 minutes on room temperature.
After ligation a transformation was performed.
To do: Mini-Prep and test digestion in order to verify assembly of beta globin and YFP. After verification of the fusion of the two fragments this construct is ready for the next BioBrick assembly step.
Comments: Test digestion looked good: No 1143 bp fragment was detectable in neither test digestion.
P158 was sent for sequencing to GATC.
Comment: Because sequencing of the right ITR delivered that perhaps a 15 bp fragment is missing in the sequence, we wanted to check, whether this fragment is also not present in the original pAAV_MCS plasmid or whether the loss of these 15 bp happened during cloning.
Comments: The test digestion showed that there're two bands between the 100 and 200 bp marker nucleotides! By comparing the gel picture with the last mini-prep digestion of the fancy method we found out that the space between the two bands are similar. Because of that we assumed that the 15 pb are already missing in the original Stratagene plasmid!!!
Conclusion: Because we already tested the Stratagene Kit via YFP expression, we can conclude that the usage of the "wrong" rigth ITR works. Therefore we decided to continue working with this "mutated" = ENGINEERED :) right ITR as BioBrick.
pSB1C3_rightITR and pSB1C3_leftITR were digested with EcoRI and PstI.
P160, P165 and P170 were send for sequencing.
HEK 293 cells were split and seeded according to the standard protocol: 2x T75 flask and 10x10cm cellculture dishes.
Results: no clones could be picked because there were no clones. Maybe the PCR purification or one of the following steps did not work (the PCR purifications were quite bad) so i purified the PCR products again yielding very low DNA concentrations (0-13,28 ng/µl) and still very obvious pollution of the sample. Nonetheless a ligation with the insert Rep40+DMSO, Rep52, Rep52+DMSO and the vector pSB1C3 was carried out following a transformation and according to the standard protocols. Hopefully there will be some clones tomorrow.
Transformation was performed with lab XL1B and iGEM XL1B cells. iGEM plate contained just 25 colonies, while lab plate contained around 50 colonies. Conclusion: the production of competent cells has to be repeated.
Quickligase was used for ligation.
Transformation was performed according to the standard protocol with DYT and BL21 cells. The cells were spread on the agar plates containing chloramphenicol.
P58 can be used for further RepCap-experiments.
HT 1080 cells were split and seeded: 3x10^6 cells per into each well of a 6-well plate.
A431 cells were washed and received new medium.
There were no clones on the agar plates so they were put back into the 37°C room. Maybe there will be some clones tomorrow.
Comments: Another approach was done because there were no clones for Rep 40 and only two for Rep 52 (Cloning see 06.08.2010). PCR was performed again with DMSO. Samples from gel extraction are stored in 4°C freezer.
PCR products were eluted with 20 µl BE puffer following the standard protocol.
Comments: mGMK was cloned into the pSB1C3 backbone in order to send it to the parts registry!. The sequence analysis revealed that the mGMK was successfully cloned into pSB1C3 plasmid. This polasmid is in the RFC25 standard.
Results sequencing: Sequence read ok!
Comment: A mutation has been found in the pSB1C3 backone which was found generally in the backbone which we received from the iGEM HQ.
The motivation: first we want to create new stock of AAV without GOI, the next viral stock is with correct TK/GMK, the last stocks are with different YFP-amounts to check if the amount of GOI is a critical step for transduction effency (transgene expression).
one 6er dish was prepared for making pictures and one T75 Flask.
Comments: In order to proceed with the BioBrick assembly the pasmids pSB1C3_left ITR and pSB1C3_CMV were used and pSB1C3_rigthITR and pSB1C3_hGH were used in order to obatin plasmids with both sequences.
The correspnding bands were cut out and Gel-Extraction was performed according to protocol.
For ligation, T4 ligase was used.
Transformation carried out according to standard protocol (BL21). Cells were plated on an agar plate with chloramphenicol.
Comments: Last week's cloning of SDM_SspI and SDM_PvuII didn't work out, so this is the repetition. In this approach the same restriction enzymes and amounts of DNA were used.
Transformation performed according to the standard protocol (BL21). The cells were plated on a agar plate with chloramphenicol.
Comments: We received the rep_cap insert ordered from Mr. Gene today. In order to used and clone the insert into the desired sequences a retrafo has to be performed.
A midi prep of the two Rep52+DMSO clones was performed yielding concentrations of 261,35 ng/µl (clone 1) and 240,47 ng/µl (clone 2). Unfortunately we noticed today that we should not use EcoRI to clone Rep52 into pSB1C3 because EcoRI cuts two times within the sequence of Rep52.
The annotations a and b were added because two midi-preps of P81, P82 and P158 were carried out.
sequencing of P184 was successful, P5 is in without TATAbox. this plasmid can be used.
P181 and P180 are thrown away because of the result of the test digestion.
Comments: Digestion of the samples will be done tomorrow with XbaI and SpeI.
Elution was done with 20 µl Pe buffer following the standard protocol.
comment: DNA was eluted into previously used tube, therefore contamination is possible.
One cut is expected (PvuII in CFP).
Comment: There are 6 bands now. This test digestion again looks very different from that of P157.2 done by Jessica (lab day 82). Results should be discussed tomorrow.
Comments: Test digestion didn't work out, three additional clones were picked from the plate we get from yesterday's Trafo to retry tomorrow.
Put in 37°C room on shaker at 20.45hours.
The motivation: In the past, the percentage of living cells in FACS was very low (~ 70%) so we want to check if the AAV-induced stress is responsible for cellular death, or the detaching procedure (trypsin and mechanical stress).
Test Digestion showed that the cloning was succesful.
Comments: In order to proceed with the BioBrick assembly the plasmids pSB1C3_leftITR_CMV was cut with SpeI and PstI, pSB1C3_betaglobin_mVenus was cut with XbaI and PstI. The insert fragment containing betaglobin_mVenus was ligated to the vector fragment containing the leftITR_CMV promotor.
The corresponding bands were cut out and Gel-Extraction was performed according to protocol.
Comments: This time digestion was done with XbaI and SpeI (means it has to be checked if the inserts are cloned into the vector in the right orientation).
For the Ligation 1µl T4 buffer (2x) and 1µl T4 ligase were used.
Comments: Because it still is unclear if SDM of pSB1C3_SspI_PvuII has worked, we decided to use the one time mutagenisised vector pSB1C3_SDM_SspI, to clone bla into it and to make the second mutation of PvuII after bla is inserted. .
Comments: The wrong enzymes were used. Experiment has to be done tomorrow.
comments: Test digestion of pSB1C3_SDM_SspI/PvuII with ClaI, BpmI and SspI looks the same as with ClaI and BpmI. Therefore we can assume that there is no additional restriction site within the vector. But still the bands above 3000 bp cannotz be explained. A new test digestion will be performed tomorrow to check the size of the vector.
The approach cut with ClaI, BpmI and PvuII looks strange. Propably, PvuII does not work correctly. New PvuII-HF was ordered and delivered so this can be verified tomorrow.
pSB1C3_RFC25_longlinker digested with SspI and PvuII seems to be not completely digested This could correspond to non-working PvuII. pSB1C3_CFP looks like expected.
comment: To verify the correct deletion of the SspI restriction site it was sent for sequenzing.
comment: pSB1C3_SDM_SspI/PvuII (P185) is empty. For plasmid production DYT + Cm was inoculated from the glycerol stock (B159).
In first place we want to create new stock of AAV without GOI, the next viral stock is with correct TK/GMK, the last stocks are with different YFP-amounts to check if the amount of GOI is a critical step for transduction effency (transgene expression).
Ten viral stocks with 3 ml each were prepared.
Gycerol stock was prepared for pMA_RC_insert and labeled B167. No glyerol stock for pSB1C3_SDM_SspI/PvuII because cells from earlier stock werer used for Mini-Prep.
Miniprep was performed according to protocol.
No test digestion was performed.
The transformation was done following the standard protocol using XL1B cells.
Comments:Yesterday the pcr-product of bla14FM was cut with the wrong enzymes, so no overhangs could result. Today it will be repeated with the rihgt ones (SpeI and NgomIV) .
gel was cut and gelextraction was performed according the qiagen standard protocol.
Plasmid was transformed into XL10-Gold cells.
By having a closer look to the BioBrick PCR we found out that the fragment sizes didn't fit to the expected sequences. In addition to that the test digestion also showed some discrepancies.
Because we weren't able to find out in which steps of the BioBrick production things went wrong, we decided to discard all referring glycerol stocks and plasmids and to perform the Rep68, Rep78 and AAP BioBrick PCR again!!!
Comment 13.08.2010 (Hanna): We wondered how the whole Rep stuff could be sequenced reverse AND forward without any forward sequencing primer... By checking the "sequencing book" we found out that always the wrong primers were used: the GOI primers can be used in order to sequence the Gene of interest = GOI (!) in pAAV_MCS and NOT the pSB1C3!!!!!!!!!
comment: In order to assemble our complete constructs mVenus has to be included into the pSB1C3_leftITR_CMV vector.
T4 ligation was performed according to protocol.
Transformation was performed according to protocol.
Plate was prepared and put in 37°C room.
In order to "save" the plasmids/BioBricks which has been prepared and confirmed for submitting it to the parts registry a new box have been prepared. The box should contain 30 µL of the desired construct cloned into the pSB1C3 vector and the BBa number given from the headquarter.
In our nomenclature plasmid excel list the constructs which we aliquoted should be highlighted in blue and commented with the given Bba number. Additionally it should be taken in consideration that enough DNA should be in the "working" plasmid box. Therefore, if necessary, inoculate DYT medium for Mini-Preps.
In this case ALL the glycerol stocks were used for incoluating 10 mL DYT medium containing 10 µL chloramphenicol respectively.
To do: Mini-Preps of the over night cultures.
We seeded cells according to standard protocols.
The YFP expression is still quite low. This was kind of anticipated, the success in this experiment is the fact, that the amount of living cells is at a very high level! (~90%). The conclusion: Detach the cells fast! Use PBS which is not at 37°C (the PBS was 25min in the water bath), transfer the cells fast on ice after detaching!
Is it possible, that freeze/thaw circles reduce the transduction efficiency?
Intention: receive enough plasmid for a sequencing and maybe futher working steps.
Comment: In order to target the EGFR receptor of our A431 cells different approaches have been figured out: Besides loop insertions, N-terminal fusion to the VP2 protein will be performed. In addition to that a VP1 insertion approach (after Grieger et al., 2007) was planned.
Fusion and insertion molecules will be e.g. His-tag, Affibody, Darpin, GFP. These approaches can be combinated with the loop insertions.
Comments: There are small and big colonies on the plate. Maybe problems with the agar plate. Picked colonies of pSB1C3_Rep40 sample 1 and pSB1C3_Rep52 sample 1 are small colonies. Picked colonies of pSB1C3_Rep40 sample 2 + 3 and pSB1C3_Rep52 sample 2 + 3 are standard size colonies.
Comments:Results look bad. Rep52 clone 3 looks good, send for sequencing.
For the Ligation 1µl T4 buffer (10x) and 1µl T4 ligase were used.
Comment: In order to target the EGFR receptor of tumor cells via Affibody or Darpin (antibody fragment is also possible!!!), or in order to expose e.g. His-Tags on the virus surface, two approaches have been figured out.
Aim of the experiment: We want to produce biobricks from Rep 68, Rep 78 and the AAP.
Gel extraction was perfomed according to protocol.
For the purification 200 µl of buffer PBI was used.
For the Ligation 1µl buffer (10x) and 1µl T4 ligase were used.
We want to knoe which confluence of HEK293 cells is optimal for AAV production.
Comments: Sequence analysis of three clones revealed that all sequences contain some mutations in the regions where the primer annealed. This was due to the extreme primer lentgh. Primers that long should be ordered as HPLC purified in order to avoid mutations in the primer sequence.
Clone 1: Mutation in the SalI site of the loop insertion site in the Viral Brick MCS lead to the removal of the recognition site. CANNOT be used for the loop insertions!!!!
Clone 2: Mutation in the region of the primer upstream of the loop insertion sites SalI and PvuII.
Clone 3: Mutation in the region of the primer downstream of the loop insertion sites SalI and PvuII.
Next steps: Two additional clones will be picked and sent for sequencing (clones 4 and 5). In order to obtain a construct without any mutations, clone 2 and clone 3 couldbe cloned together. Each clone will be digested with SalI and BamHI. Clone 2 will be the "insert", clone 3 is going to be the "vector". Therefore the fragments will (hopefully) not contain anymore mutations in the relevant sequence.
Comment: The plamsid numbers in the picture (P179-P184) correspond to the glycerol stocks B179-B184. The corresponding plasmid numbers are: P208-P213.
Results: Two (three) different approaches can be seen on the agarose gel.
P178-P181 (blue): The plasmid pSB1C3_leftITR_CMV_betaglobin_mVenus was digested with NgoMIV and EcoRI. Expected bands were: 2873bp and 1343 . As it can be seen in the picture, the two fragments ran too high (~300bp). As it is almost normal for the pSB1C3 plasmid, we should figure out why this is always the problem for this vector.
P182-P183 (red): The plasmid pMA_RepCap_SDM_insPvuII was digested with PvuII as well as the control vetor pMA_RepCap where no site-directed mutagenesis was performed in order to see the difference. As it can be seen in the picture above, the expected band sizes of 263bp,1384bp and 2133bp correspond to the bands detected in the gel. The control vector instead reveals only two bands detectable. Therefore site-directed mutagenesis was successfully performed.
P177 (green): pSB1C3_hTERT. For further details see extra part below (Test digestion of pSB1C3_hTERT.
Test digestion: or test digestion, pSB1C3_leftITR_CMV (P188) was digested as well.
comment: Through digestion, fragments would have similar sizes. Therefore, a new digestion will be performed tomorrow.
Comments: pSB1C3_hTERT was sent for sequencing and revealed that the PCR and the inserting of the PCR product : promoter for hTERT was sucessfully performed. But the sequencing results showed a new AgeI restriciton site in the plasmid backbone. We could not see if the new recognition site was due to the bad sequence read or that there is really a new AgeI site. Therefore, a test digestion will be perfomed with PstI and AgeI in order to figure our wheter AgeI is present in the backbone or not.
After the incubation,the samples were loaded on the 1% agarose gel, and run at 110 V for 45 minutes.
Results: As it can be seen in the sequencing results of pSB1C3_hTERT there could have been an additional AgeI site in the vector. If AgeI would have been in the vector, two fragments have been expected by digetsing the plasmid with AgeI and PstI. As it can be seen in the agarose gel, lane 11 (labeled with the green P177) only reveals one lane instead of two. Therefore it can be assumed that NO AgeI site is in the pSB1C3 backbone.
15 Clones were picked saturday night so that a mini-prep could be performed on sunday.
Sequence assembly showed that the suffix of the Rep protein 52 is ok and that PCR worked well.
There can be seen that a silent mutation from AGG to AGA was inserted, but it will have no effect on the protein because codon usage in H. sapiens is nearly the same for both codons.
The prefix could not be analysed because of bad sequence read quality. For further verification, another sequencing with the forward primer has to be performed.
Next steps: Send plasmid for another sequencing round with forward primer in order to analyse the prefix.
No test digestion for pSB1C3_SDM_SspI_Bla14FM clone 4 and 5.
For test digestion, pSB1C3_leftITR_CMV_mVenus (P206-207) and pSB1C3_leftITR_CMV (P188) was digested as well.
Samples P208-220 were digested with XbaI and SpeI.
Samples P207, P207 and P188 were digested with XbaI and NgoMIV.
comment: Rep- and AAP approaches have to be repeated. We did not realize that the vector can religate! Plasmids and glycerol stocks were thrown away.
One colony was picked from agar plate and dispersed into 10 ml DYT+ Amp and incubated over night @ 37 C.
Sequencing showed that clone 2 had a mutation in the vector, clone 3 had one in the BLA sequence. Therefore, both were cut with BamHI and SalI, then the BLA fragment of clone 2 and the vector fragment of clone 3 were ligated. The final construct shouldn't contain any more mutations.
Two additional Clones were picked (clones 4 & 5) and sent for sequencing, labeled: iGEM4.1 and iGEM4.2.
The fluorescence was quite low (~ 5%)in nearly every well! The 40µg YFP wells were the only exception.
Remember: This was just a qualitative check of our constructs! The scale bar is missing but its not that dramatic. As you can see, our construct works! It is possible to kill tumor cells with our vector.
Motivation: the desired gen contains 2 iGEM restrictions sites, thus in oder to use it for further cloning, these site have to be deleted. The first SDM was performed on PstI restriction site.
Transformation was performed according to the standard protocol.
40 ml DYT was prepared with 40 µl Ampicillin and inoculated with B24 and B34, both were incubated over night in 37°C room.
Comments: Incubation at 25° after addition of SwaI for 1,5 hours. Then incubation at 60° after addition of BstEII for 1,5 hours.
Bands are really weak, tomorrow I`ll take more DNA in sample.
Aim of the experiment: We want to produce biobricks from Rep40, Rep68, Rep78 and AAP.
Comments: I forgot to dilute Rep68. Therefore, a new PCR will have to be performed tonight.
Comment: The CMV promoter is often used in combination with a SV40 terminator. Because we want to use this promoter in the context of the VP2 fusion and VP1 insertion approaches, oligos were designed in order to produce a SV40 terminator BioBrick (from the pEGFP-C1 plasmid).
Comment: In order to perform the VP2 N-terminal fusion and VP1 insertion approaches, we decided to use pEGFP-C1 as expression plasmid. This plasmid contains an EGFP under the control of the CMV promoter and the SV40 terminator. In order to exchange the EGFP with our targeting constructs a PCR of the CMV_backbone_SV40Terminator should be performed. The referring primers were designed with EcoRI and PstI restriction sites. One should keep in mind that this expression plasmid mustn't digested with NgoMIV and it contains Kanamycin-resistance!
Comments: In order to obtain a overview (more or less plausible ;-)) I sorted out how the final plasmid need to be constructed. In the picture you can see the most important plasmids which we will need for the retargeting approaches starting tomorrow.
Sequencing results: there is no BLA in clone 4. Clone 5 showed a flawless BLA so it will be used for the next site directed mutagenesis.
Further procedure including the transformation was performed according to the standard protocol.
pMA_RC (P222) sent for sequencing with O41 (Cap 4000 for) to check if the EaglII restiction site was successfully changed to PvuII.
pAAV_RC (P158) sent for sequencing with GATC_std_SK, Rep_1250 for (O35), GATC_std_UP-2 to check if all deleted restriction sites are present (PstI, BamHI, SalI).
Aim of the experiment: We want to produce biobricks from Rep 68.
comment: Elution was put in 4°C room. Work will be continued tomorrow.
This construct without the hGH polyadenylation sequence will be used to determine wether/how much hGH affects translation of the virus gene.
Clones were inoculated in 10 mL DYT medium containing 10 µL of antibiotics and were put in 37°C room on shaker.


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America and Saudi Women Again!
By Sheikha Al Dosary
Before President Obama visited Saudi Arabia this last weekend, he was urged by 70 members of Congress who signed a bipartisan letter to raise critical human rights cases in Saudi Arabia. They also advised him to meet with Saudi female activists.
Amnesty International also asked him to select a female Secret Service officer to drive his car in Saudi Arabia and meet Saudi Arabian women who protested the driving ban.
All this urging seemed to have no effect, not because he didn't believe in those issues, but considering all critical political issues in the region, the ban on Saudi women driving is nothing in comparison.
The US administration's attitude regarding this issue has varied over the years. In 2013 Secretary of State John Kerry, when asked about Saudi women who are demanding the right to drive, said: "It's no secret that in the United States of America we embrace equality for everybody, regardless of gender, race, or any other qualification, but it's up to Saudi Arabia to make its own decisions about its own social structure. … There's a healthy debate in Saudi Arabia about this issue, but I think that debate is best left to Saudi Arabia."
That was a well-received answer by many Saudis, which portrays a totally different attitude than former Secretary of State Hillary Clinton, when she said she supported the ban's repeal, adding that the ban is "hard to even rationalize in today's world."
Although I personally support her, I'm against the US interfering in social policies in Saudi Arabia. Why do Americans have to interfere with Saudi women's lives, or in any women's lives other than American women? Is our life here in the US as women so perfect that we reached the satisfaction level to start working on other women's issues?
President Obama has chosen instead to honor and highlight the work of successful Saudi woman during his trip. He honored activist Maha Al-Muneef with the International Woman of Courage Award, for her role in drafting and advising the first law ever to protect women in the Kingdom from abuse and criminalized domestic violence.
President Obama Presents the International Women of Courage Award to Dr.Maha Al Muneef
This action sends a powerful message about his foreign policy toward Saudi women's issues and Muslim women in general.
Yet this was not the first attempt to integrate concerns about Saudi women's rights into US foreign policy. In 1991, many American civil rights activists lobbied to support Saudi women's rights and defy the ban on driving. This urging was based on the experience of several deployed female American military personnel and the accounts of female journalists who witnessed the ban firsthand. Despite their efforts, nothing has changed.
The reason, according to Kelly J Shannon's study I'm glad I'm Not a Saudi Woman: the First Gulf War and US Encounters With Saudi Gender Relations, is that women's rights were not at the top of President George Bush's agenda at the time, who had close personal ties to the Saudi monarchy At that time, America chose not to condemn "gender apartheid" in Saudi Arabia, despite all of the calls for change.
The sad reality since that incident is that Saudi women have been isolated from important decisions being made about them in a foreign country. The US is speaking out on Saudi women's behalf, which is not what Saudi women want. What happened to democracy here?
After analyzing social media campaigns, as well as the views of Saudi columnists and writers for the past week and earlier, many Saudis refuse what they view as America's interference in their internal affairs.
At the same time, many Saudis from various backgrounds show their support for the Saudi women driving campaign that has been established since 2011. Saudi2drive campaign's followers increase, as well as women getting behind the wheel and driving, regardless of the penalties. Detention at the police station and signing pledges not to do it again are no longer effective deterrents. Since the lunch of another campaign from October 26 last year, more than 100 women have gotten behind the wheel, and almost every other day there is a new video of a Saudi woman driving. https://twitter.com/oct26driving
Petitioning Saudi Government to Lift the Ban on Women Driving https://www.youtube.com/watch?v=Hwz6RMGntUQ
Although King Abdullah initially approved of this step, he give Saudi society the final say on this decision. Saudi Writer Madawi Al Rasheed said the issue is meant to divide Saudis and keep them busy. "It distracts Saudis from more urgent questions such as political representation, civil and human rights and unemployment among both men and women."
It is worth mentioning that Saudi women have been driving in villages and the outskirts of big cities without repercussions for some time. In October 1987, photographer Jodi Cobb captured this for the National Geographic magazine article Women of Saudi Arabia. She confirmed that Bedouin women enjoy greater freedom of dress and movement. Driving is a privilege denied to women in the city. In her photos she captured a woman driving her family's water truck.
If Bedouin women drove a long time ago, could we witness their Saudi counterparts driving in cities soon? And most importantly, could we witness more Americans honoring and celebrating successful Saudi women who work behind the scene for progressive change?
(Sheikha Aldosary is a freelance journalist covers social issues and women rights in the Gulf region)
Barack Obama arrives in Saudi Arabia for brief visit with upset Arab ally http://www.theguardian.com/world/2014/mar/28/barack-obama-saudi-arabia-arab-ally
http://www.amnestyusa.org/news/press-releases/amnesty-international-calls-on-president-obama-to-bring-a-female-secret-service-driver-to-saudi-arab
http://www.al-monitor.com/pulse/originals/2014/03/saudi-obama-human-rights-congress.html
Letter to President Obama about human rights in Saudi Arabia http://backchannel.al-monitor.com/wp-content/uploads/2014/03/FRANKS-MCGOVERN-WOLF-SPEIER-Letter-to-President-Obama-about-Human-Rights-in-Saudi-Arabia.pdf
Kerry Takes Softer Line Than Hillary on Saudi Women Drivers
http://swampland.time.com/2013/11/05/kerry-takes-softer-line-than-hillary-on-saudi-women-drivers/
President Obama Presents the International Women of Courage Award to Maha Al Muneef
http://www.whitehouse.gov/photos-and-video/video/2014/03/29/president-obama-presents-international-women-courage-award
Al-Rasheed: The Saudi Duality on Women
http://www.al-monitor.com/pulse/tr/originals/2013/09/saudi-women-drive-viral-video-cleric.html
President Obama reportedly to meet with female activist in Saudi Arabia on day when women have pledged to defy driving ban
Saudi Arabia's oppression of women goes way beyond its ban on driving
Saudi Cleric Confirms That Being a Woman in Saudi Arabia Sucks, Big Time
Saudi women drive with few problems
Saudi Arabia 'rethinking women driver ban'
Saudi Arabian women raise their voices
Saudi Women Defy Driving Ban
Woman arrested for driving in Saudi Arabia
Managing Writer
Managing Writer at The Arab Daily News online
The Arab Daily News is the newspaper of record for the Arab American Community, and publishes and writes original articles on news, features, commentary, business, politics, sports, entertainment and more. Managing Writer of the Arab Daily News, reviews and posts articles for the journalists and bloggers who submit their writings for this popular online newspaper newspaper.
Latest posts by Managing Writer (see all)
African Author Mohammad Elsanour releases new novel "I Can't breathe" - January 14, 2021
Last Lunch—Reflecting on Aljazeera's Documentary of Yemen's President Al-Hamdi - October 21, 2020
The Novel Coronavirus is a Challenge to All of Us - April 7, 2020
April 2, 2014 Managing Writer
Arab World, Bloggers, Commentary, Gulf StatesBarack Obama , International Women of Courage Award , John Kerry , Madawi Al Rasheed , National Geographic , Saudi , Saudi Arabia , United States
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redpajama_set_name: RedPajamaCommonCrawl


sustainability and conservation for our clients and ourselves.
Comply with all relevant national and regional environmental legislation in every country in which XTG operates.
Ensure that the XTG's products and services create the lowest possible impact on the environment.
Contribute the maximum possible economically to local communities, particularly to the green economy, with purchases of ecologically friendly products, locally (preferably organically) grown produce, foodstuffs and beverages(green procurement).
Provide training to promote awareness of environmental issues, sustainability and good practice, particularly those related to the XTG's activities and services.
Make all those who work with XTG aware of their performance requirements within its management processes to ensure continually improved environmental practice.
Provide the framework, involving all those who work with XTG, for reviewing all environmental objectives and targets on a regular basis.


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Lawrence Klausman Parks
Olathe, Kansas, USA
Fringe player in numerous B productions of the 1940s who came to prominence as the lead in "The Al Jolson Story" (1946). Parks' career was curtailed when he admitted his past Communist affiliations to the House Committee on Un-American Activities. Married to actress Betty Garrett from 1944....
AC/DC (1969)
Freud (1962)
Dr. Joseph Breuer
Cross-Up (1958)
John Desmond
Love Is Better Than Ever (1952)
Jud Parker
Emergency Wedding (1950)
Peter Judson Kirk, Jr.
Jolson Sings Again (1950)
The Jolson Story - Movie Poster
Counter-Attack (1945) -- (Movie Clip) Too Much Courage
Jolson Story, The - (Original Trailer)
Reveille With Beverly - (Television Trailer)
Down To Earth (1947) -- (Movie Clip) He Just Doesn't Like Me
Betty Garrett
Actor. Married from September 8, 1944 until his death; mother of Parks's two sons.
Fringe player in numerous B productions of the 1940s who came to prominence as the lead in "The Al Jolson Story" (1946). Parks' career was curtailed when he admitted his past Communist affiliations to the House Committee on Un-American Activities. Married to actress Betty Garrett from 1944.
The Gallant Blade (1948)
Lt. David Picard
Down to Earth (1947)
Danny Miller
The Swordsman (1947)
Alexander MacArden, also known as Donald Fraser
The Jolson Story (1947)
Al Jolson [assumed name of Asa Yoelson]
Her Husband's Affairs (1947)
Renegades (1946)
Ben Taylor, alias of Ben Dembrow
Counter-Attack (1945)
Kirichenko
She's a Sweetheart (1944)
Stars on Parade (1944)
Danny Davis
Hey, Rookie (1944)
Jim Lighter
Sergeant Mike (1944)
Pvt. Tom Allen
The Black Parachute (1944)
Michael Kaligor Lindley
The Racket Man (1944)
Larry Lake
First Comes Courage (1943)
Reveille with Beverly (1943)
Power of the Press (1943)
Jerry Purvis
Deerslayer (1943)
Jingo-Good
Is Everybody Happy? (1943)
Redhead from Manhattan (1943)
Destroyer (1943)
Ensign Johnson
Blondie Goes to College (1942)
Rusty Bryant
Submarine Raider (1942)
Sparksie
Alias Boston Blackie (1942)
Joe Trilby
Flight Lieutenant (1942)
Sandy Roth
You Were Never Lovelier (1942)
A Man's World (1942)
[Charles] Chick O'Driscoll
Hello Annapolis (1942)
The Boogie Man Will Get You (1942)
Bill Layden
Canal Zone (1942)
Atlantic Convoy (1942)
They All Kissed the Bride (1942)
Joe Krim
North of the Rockies (1942)
You Belong to Me (1941)
Harvard, Here I Come (1941)
Eddie Spellman
Harmon of Michigan (1941)
Sing for Your Supper (1941)
Mystery Ship (1941)
Three Girls About Town (1941)
Simon Birch (1998)
9 1/2 Weeks (1986)
Sound (Feature Film)
Hot Line (1970)
A Bride for Brenda (1969)
Hot Kiss (1969)
Music (TV Mini-Series)
The Brave Little Toaster Goes to Mars (1999)
Feature acting debut "Mystery Ship"
Became famous for playing Al Jolson in "The Jolson Story"
Last feature appearance as Dr. Joseph Breuer in John Huston's "Freud"
Counter-Attack (1945) -- (Movie Clip) Too Much Courage Paul Muni as Alexei leads the band of Russian military engineers arrived to a key Eastern Front theater, bragging recklessly until the commander Kostyuk (Roman Bohnen) demands their attention, revealing his plans to surprise the Germans, Marguerite Chapman as comrade Lisa, in director Zoltan Korda's Counter-Attack, 1945.
Down To Earth (1947) -- (Movie Clip) He Just Doesn't Like Me Rita Hayworth is posing as actress "Kitty," really the goddess Terpsichore, persuading director Danny (Larry Parks) to make their show fancier, over the objections of his pal Eddie (Marc Platt) in the much-later musical sequel to Here Comes Mr. Jordan, 1941, Down To Earth, 1947.
Love Is Better Than Ever -- (Movie Clip) Long Beach, California Agent Jud (Larry Parks) is determined to prove he's no longer stuck-on dance instructor Staci (Elizabeth Taylor), who bamboozles him again then performs with about 200 kids, in Love Is Better Than Ever 1952.
Love Is Better Than Ever -- (Movie Clip) Gene Kelly Big-time agent Jud (Larry Parks) introduces Staci (Elizabeth Taylor), the pretty dance instructor from New Haven, to the real Gene Kelly, at "21," part of a night on the town in Love Is Better Than Ever, 1952.
Counter-Attack - (Original Trailer) A Russian officer (Paul Muni) is trapped in a bombed building with seven Nazi soldiers in Counter-Attack (1945).
Jolson Story, The - (Original Trailer) Larry Parks stars as the famous blackface performer of stage and screen, Al Jolson, in The Jolson Story (1947).
Reveille With Beverly - (Television Trailer) Frank Sinatra, Duke Ellington and Count Basie are among the delights in the wartime musical Reveille With Beverly (1943).
Jolson Sings Again - (Original Trailer) World War II brings Al Jolson (Larry Parks) out of retirement in Jolson Sings Again (1950).
Andrew Parks
Actor.
Garrett Parks


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Q: How to use winfo_children to modify the tkinter buttons gridded in different frames? I got  a list of all the buttons and the frames where they extend from using this code:
def childrens (window) :
    lst = window.winfo_children()

    for item in lst :
        if item.winfo_children() :
            _list.extend(item.winfo_children())

    print (_list)

which gives me this:
[<tkinter.LabelFrame object .!labelframe>, <tkinter.LabelFrame object .!labelframe2>,
 <tkinter.LabelFrame object .!labelframe3>, <tkinter.LabelFrame object .!labelframe4>,
 <tkinter.LabelFrame object .!labelframe5>, <tkinter.LabelFrame object .!labelframe6>,
 <tkinter.Button object .!labelframe.!button>, <tkinter.Button object .!labelframe.!button2>,
 <tkinter.Button object .!labelframe.!button3>, <tkinter.Button object .!labelframe.!button4>,
 <tkinter.Button object .!labelframe2.!button>, <tkinter.Button object .!labelframe2.!button2>,
 <tkinter.Button object .!labelframe2.!button3>, <tkinter.Button object .!labelframe2.!button4>]

Now how do I really get to use this to modify the buttons that I implemented?

A: The items in the list are widgets. You can directly iterate over them and do whatever you want.
There are bugs in the code you posted, but assuming those bugs were fixed you could directly iterate over the contents of _list or list (it's not clear what you intended). You can use isinstance to determine if the widget is a button.
for widget in _list:
    if isinstance(widget, tk.Button):
        widget.configure(...)

redpajama_set_name: RedPajamaStackExchange


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Atlanta airport mess: How does this happen?
The power is back on at the busiest airport in the world.But as passengers scramble to make rescheduled flight...
Posted: Dec 19, 2017 11:13 AM
Updated: Dec 19, 2017 11:13 AM
The power is back on at the busiest airport in the world.
But as passengers scramble to make rescheduled flights during the busy holiday season, the question remains: Why did Atlanta's Hartsfield-Jackson airport go dark for nearly 11 hours?
The short answer is that an electrical fire in a tunnel beneath the airport crippled both the main power system and its backup. Experts say the broader issue is that America's airports don't seem to be prepared for such events.
Georgia Power, the utility that provides Hartsfield-Jackson with electricity, said in a statement Monday that "a piece of Georgia Power switchgear located in an underground electrical facility could have failed and started a fire."
Because the fire was in a tunnel "adjacent" to the backup cables, the fail-safe system was knocked out, too, Georgia Power said.
Workers then had to wait between an hour and a half and two hours to enter the tunnel and start repairs due to safety concerns, Atlanta Mayor Kasim Reed said at a press conference on Sunday.
Related: Thousands trying to get out of Atlanta after lights went out at Hartsfield airport
"Because of the types of fumes that came through the tunnel as a result of the fire, we had to take some time before the team from Georgia Power could access the tunnels," Reed said.
Experts who study transportation infrastructure, however, expressed disbelief that such an incident could knock the Atlanta airport's power out for so long.
"[A] fire shouldn't paralyze the country's busiest airport for half the day. It may take a couple hours to bring alternative power to the airport, but if the airport infrastructure itself was undamaged, it shouldn't have been nearly as painful," said Joseph Schwieterman, a public policy professor at DePaul University who specializes in transportation and urban planning.
Georgia Power's investigation into exactly what happened will last the rest of the week, CEO Paul Bowers said Monday on "Good Morning America."
For air travel, it's been "death by a thousand cuts," Schwieterman said. From bad weather to computer issues to Sunday's power outage, airlines have been repeatedly hit with shutdowns, exposing a number of vulnerabilities in the process.
Severe thunderstorms in Atlanta in April seriously disrupted Delta's operations for days. British Airways had a "major IT system failure" in May that interrupted flight schedules worldwide. And in August 2016, Delta experienced a computer outage that downed critical systems for nearly five hours.
"Airport outages seem to be happening more and more, for a number of reasons, but planning for these outages is not prioritized," said Megan Ryerson, a professor at the University of Pennsylvania who studies airport infrastructure planning.. " ... It's something I think requires a lot of focus."
According to Ryerson, carriers and airports should put more energy into ensuring day-to-day operations are "as reliable and predictable as possible, rather than focusing on growth."
Since top U.S. airlines rely on certain cities as hubs, the ripple effect from some airports can be particularly severe. The primary hub for Delta is in Atlanta, while United's is in Chicago and American's is in Dallas.
Sunday's fire and subsequent power outage caused 1,180 flight cancellations to and from Hartsfield-Jackson airport that day alone. Hundreds more were announced on Monday.
"As a country, we're dependent on just a few critical airports. And when one of them goes out, it's devastating," Ryerson said.
Atlanta's Hartsfield-Jackson airport restores power after crippling outage
Atlanta airport outage complicates insanely busy holiday travel
Will Atlanta's airport outage hurt its Amazon HQ2 bid?
Delta wants its money back after Atlanta airport blackout
Atlanta airport is now offering flights to Wakanda -- sort of
Part of Delta plane catches fire at Atlanta Airport
CBP officers protect and save lives at Atlanta airport
Another Wells Fargo mess: Pet insurance ripoff
Sheriff: Don't mess with the Uber driver


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Oscar's CrypTEA is portable file and folder encryption tool from Mediachance.
There are 2 editions of this application: free and paid.
The free version is for personal use only, but is reasonably limited and very useful in everyday work.
CrypTEA is easy to use, with clear interface. The folder and its subfolders will be encrypted or decrypted in their place. File names are encrypted too. This light, portable tool (single .exe file about 5.2 MB) is safe for remote access and may work with files on NAS storages.
How to extract: Download the ZIP package and extract to a folder of your choice. Launch APPNAME.EXE.
Double-entry for CrypTEA: see http://www.portablefreeware.com/index.php?id=2815.


ZOCCO, ZOCLE, ZOCCOLO, n. [L., a sock.] A square body under the base of a pedestal, &c. Serving for the support of a bust, statue or column.
A square body under the base of a pedestal, &c., serving for the support of a bust, statue or column. – Cyc.
ZOCCO, ZOCLE, ZOCCOLO, noun [Latin , a sock.] A square body under the base of a pedestal, etc. Serving for the support of a bust, statue or column.
INCAU'TIOUSNESS, n. Want of caution; unwariness; want of foresight.


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Sometimes a simple color change is all you need to turn something into exactly what you want. This custom wedding garter design is a customized version of garter style 353 from my collection. The bride wanted to glam it up even more and with black lace. I think it turned out gorgeous and very sophisticated! This custom wedding garter is an ivory satin background with a black lace overlay and a black satin bow (2 inches wide).
To see more designs, visit my custom garter gallery or check out the custom garter design blog series.


From the thousand photographs online concerning 12X18 Frame, picks the very best collections using ideal quality simply for you all, and this photographs is actually considered one of images collections in our ideal images gallery concerning 12X18 Frame. I am hoping you can as it.
This particular impression (Picture Frame Bincentart throughout NewestFrame 1412 32) preceding can be classed using:posted by simply @Wadeframes.com on July, 11 2018. To find out most images with 12X18 Frame images gallery please adhere to 12X18 Frame.


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The Top 5 Cars with the Coolest Built-in Technology
by Michael Thompson | Apr 14, 2016 | Used Cars | 0 comments
Car tech isn't just Bluetooth. Here are the cars with the coolest technology.
Go to your favorite (other than us!) major automotive news website, and check out the top stories. I guarantee the top story is about connected cars. Currently, it's the hottest topic in automotive news. But what does it mean, and where can you find a connected car?
Just ten years ago connected technology meant that your car had a Bluetooth system. Today it means parking sensors, front and rear cameras, adaptive lane control and lasers that monitor distance to the car in front of you. Connectivity isn't just safety features, it still means having a car with 4G WiFi and the ability to integrate your phone seamlessly.
I went ahead and did the heavy lifting for you and found the top five connected used cars today. With the rapid technology advancement today I'm sure this list might be different by next year, but if you are on the market for a used car today and connectivity is important to you, start here.
This Tesla Model S is out of Lewisville, TX. With 20,000 miles, it goes for a little less than $60,000.
The Tesla Model S is the most advanced car in the connected car arena and you can currently get a top-of-the-line used model at a good discount off the MSRP. The Tesla Model S is one of the best choices because it is so user friendly that you won't even recognize that the car is doing most of the driving for you. It all starts with the huge (17-inch) tablet display in the center console that you can use to control the whole car experience. You can adjust ride height, music, temperature, surf the web, make an appointment, make a phone call and use google maps all from the giant tablet display. The Tesla Model S goes a step further and now has "self-driving" modes, which allow the driver to remove their hands up to six minutes from the steering wheel.
This Infiniti Q50 is out of Libertyville, IL. With 17,000 miles, it goes for $25,000.
Infiniti Q50S
The Infiniti Q50S is known as the first car on the road with the a "drive-by-wire" capability. "Drive-by-wire" means that there are no direct connection between the steering wheel and the wheels, just electrical signals. The Infiniti Q50S is packed with safety technology and features: it has, take a deep breath, automatic collision notification, multi-view parking cameras, front and rear parking sensors, adaptive cruise control, blind-spot/lane departure warning, forward/back-up collision warning systems, and active lane control. If you are a car enthusiast you might want to look elsewhere, because of the lack of steering feel and control. If you don't mind what a car's steering feels like, and want the car to do all the work, we just found you your next car.
This Mercedes-Benz S-Class is out of Lawrenceville, NJ. With just 8,000 miles, it goes for $94,000.
Mercedes-Benz was one of the first companies to take the "drive" into the connected car world when it adapted multiple safety technologies to the S-Class. Mercedes offers several autonomous features such as Distronic Plus with Steering, Active Blind Spot Assist, Active Lane-Keeping Assist and Collision Prevention Assist with Adaptive Brake Assist (so many assists it makes you wonder what the driver should do?), some of these features are even standard on new S-Class models. The best feature would have to be Traffic-jam Assist, it will safely control your car for you in traffic up to speeds of 37mph. If you have the funds to pick up a used 2014 Mercedes Benz S-Class it packs tons of safety, connected technology, and style in an excellent package.
This Audi A3 out of Riverside,CA is a steal with 6,000 miles at $30,000.
Audi has always been building safe, and fun cars but have been behind when it came to infotainment and connectivity. Audi changed that perspective when it launched their entry level Audi A3, which was focused at a younger (millennial) audience. The A3 comes with AT&T's 4G LTE network capability, and the ability to link up eight devices to that network. You could run a mobile office out of your A3, and look good while doing it. Audi allows you to stream the WiFi through the MMI infotainment system and that gives you the capability to connect to Facebook, Twitter, and 3D Google Earth Maps, just don't do all that while driving. The Audi A3 would be the perfect car for the young professional, getting their first job and wants to stay connected, while being stylish.
This Chevrolet Cruze in Moosic, PA under $15,000.
The reason the Chevrolet Cruze is on this list is its price, it's the most affordable option of the five cars listed here and it comes with 4G LTE WiFi. Who doesn't like a bargain? Chevrolet has made a big push to put 4G LTE WiFi in its entire lineup and that means even the cheap cars get it. Hurray! The Chevrolet Cruze is not the sexiest car of the five, but it's definitely the best bargain and that has to count for something, right?
If you go back twenty years ago, the only thing connecting you to your car where your hands. Flash forward to today and it seems like cars soon won't even need steering wheels. There has been a fast advancement of connected technologies in automobiles, that means that you can get cutting edge technology for bargain basement prices once the first owner has taken the depreciation hit. Which connected car would you like to take home?
Like one of these cars but don't live in those areas? Don't forget you can ship cars with UShip. Find instructions here.
Weekly News Round-Up: Friends Don't Let Friends Snapchat and Drive
#TBT: Gary Numan's "Cars"
Volvo Goes High Tech: Integrated Spotify, Keys On Your Phone
Samsung, Verizon, and the Dawn of the Connected Car


text:
Living in San Pedro Belize
Yesterday's post made me think of things that are totally new to me in Belize but now a daily part of my life. How did I live without this stuff? Simple answer…I didn't really cook (or clean for that matter). Manhattan was takeaway sushi, eating out most nights, delivered Italian food at 11pm andKeep reading »
This is the #1 question that I get asked. And I can't give you the exact figure that will work for you, but I can tell you how much it costs me to live on Ambergris Caye. Keep in mind: Ambergris Caye is probably the most expensive part of Belize. And also remember $1USD = $2Keep reading »
When moving to a new country (particularly the tropics), you have some tough decisions. I came to Belize two times in my first few months…both times with two large over- packed suitcases…both suitcases dangerously teetering on the 50lbs limit. Everything else was sold at a garage sale. But now that I look back, there are things thatKeep reading »
So…I had my work permit, was managing a popular bar/restaurant in town. Time to become permanent. $2000bzd a year for a work permit is a big expense and getting permanent resident is a life-long work permit, a one time expense. But applying for permanent residency in Belize is more of a waiting game than anything else. There areKeep reading »
Ok…so where did I leave off…I was single (still am), 32, with no kids. I put everything in storage, sold my car and moved to San Pedro, Belize in early 2007. I was ready to take a year off and enjoy my new country. I had met lots of Brits, Canadians and Americans who had moved here. Keep reading »
I've actually been getting a bunch of emails from all over the world asking me a few questions: How did I pick Belize? How easy was it to get a job? Why have I stayed? What do I love and what would I change? Well, as you can imagine, this is a long and tediousKeep reading »
Source: www.sanpedroscoop.com
Belize-Gringo Expats
Living in Belize
OUTREACH LIVING WORD CHURCH SAN PEDRO TOWN BELIZE
Thomas Vincent Ramos (17 September 1887 - 14 November 1955) was a Belizean civil rights activist who promoted the interests of his Garifuna people, and is now considered a national hero.
T.V. Ramos was born in Puerto Cortez, Honduras, on September 17, 1887 and was...
Harrison Dwith Róchez (born 29 November 1983) is a Belizean football midfielder, who plays for Platense in the Liga Nacional de Honduras.
Róchez played for several local sides in his native Belize, before moving to play in Honduras in 2007.
He made his debut for...
Population of San Pedro Belize
Pedro Town is the major settlement on the island. San Pedro s populace has grown to 10, plus year-round inhabitants, with…
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Business cards for a Philly restaurant pirate. More photos over on the Moontree facebook page.
Some photos of the cherry pies I made from my neighbor's tree. You can see a few more photos here, hope everyone has a safe and happy July 4th!
I'm falling a little behind on my blog these days! I'll try to be better, but for more frequent updates please check out my Facebook page.
With the warm weather on the horizon, here is a new wedding style under the Nature Collection. Its called Woodlands, here are some more shots – let me know what you think – thanks!


For 15 years Virginia King has lived in her house on Forrest Drive in Ridgeland, just off of Bees Creek Road. Almost every day her five grandchildren visit her and she'll even jokingly fuss that they visit too often.
The grandchildren make her happy. Last week she was visited by some other families' children, teenagers from the Salkehatchie Summer Service who helped repair her home.
The bathroom, hallway and kitchen floors needed fixing and there was plumbing improvements, but King was all smiles as she sat on a couch in her living room last Thursday.
"Oh, thank God for them," said King, who grew up in a home on Main Street. "I love them."
Across the street more teens from the Summer Service, hosted by Ridgeland's St. Paul's United Methodist Church, and several from Jasper's Boys & Girls Club were repairing Mary Williams' home.
Williams has been living there more than 20 years and for the past two years has seen water leak through her roof.
But last week the roof was repaired, led by 17-year-old Breanna Williford of Chester.
"I'm happy for them being here," Williams said.
The church has hosted the camp for two years. The teenagers are from throughout the state, including Greenville and Simpsonville. They began work early last Monday morning, stayed at night at Thomas Heyward Academy's gymnasium, and planned to finish last Friday. Food was supplied by Duke's BBQ, KFC, Jasper's Porch and Country Morning Bakery.
The Salkehatchie camps promote the word of God and also help those in need.
Kaitlin Yenson, 15, participated in her first Salkehatchie camp. A Greenville native who attends church in Simpsonville, Yenson has done other church mission work and last week she painted, sawed a pipe and tore up King's hallway, kitchen and bathroom floors. She used some dormant arm muscles, but said the experience was "magical."
"I just enjoy helping people," Yenson said. "It's really cool."
King suffers from arthritis and said it has been hard for her to walk around. There were dips in her floors and she was reluctant to walk too much. The floor by the washing machine sank 2 inches.
The summer camp teens helped fix that problem.
"You can't find nobody better than them," said King, whose grandchildren attend the public schools and range from third to 12th grade. "They've done a beautiful job. I can walk around good, don't have to worry about falling."
Yenson's brother, Andrew, also worked on King's home.
It was his first time in Jasper County, but he's done similar camps in Sumter and Aiken.
He said he does as many mission trips as he can.
"You grow spiritually within yourself," he said.
Andrew Yenson said the experiences help him realize how fortunate he is and makes him want to help others.
"Coming here and realizing the floor boards were all rotten, I don't live like that, how were they getting by? It's a realization that there's stuff out there for you to do," he said.
He left his two prior Salkehatchie camps not having entirely completed what was planned, but early last Thursday Andrew Yenson thought the group's goals would be accomplished.
Williams stood outside her home with a smile as she watched her roof and side deck be fixed.
Williford has been part of the camp for three years and this was her second consecutive visit to Jasper County.
It was hot on the roof, she said, but major work was needed. There were rotted boards and holes in the roof, but by Thursday the job was completed.
"I feel like these people need help," she said. "I have been blessed my entire life with shelter, food and these people deserve it. It's satisfying."
Williams, who has two grandchildren in Jasper County's public schools and two in Beaufort County's schools, said she's happy with the deck's repairs and is thrilled with the teenagers' kindness.
"They are so friendly," she said.
Williford said late rain on Wednesday posed a challenge to the roof, but the group was determined: "I was like, 'Let's do it, let's do it,' " she said.
Overall, teens fixed three houses last week and Williford said the experience was worthwhile.
"I've been blessed to do it," she said.


text:
Wydarzenia

 12 maja – Stefan I został papieżem.

Zmarli
 Orygenes, filozof i teolog chrześcijański torturowany w więzieniu

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redpajama_set_name: RedPajamaWikipedia


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Q: Why does app launcher icon remain in the status bar (Android 4.4.2)? On android 4.4.2 when my app starts its launcher icon appears in the status bar and remains there for the duration of the main activity, that is until finish() is not called. Is there a way not to have it appeared in the status bar? (I am using GCM notifications and one arrives having two nearly identical icons next to each other is quite confusing, needless to say that it takes unnecessary space). Note it does not happen in the previous android versions. Below is my AndroidManifest.xml.
Thanks for any advice.
<?xml version="1.0" encoding="utf-8"?>

<manifest xmlns:android="http://schemas.android.com/apk/res/android"
    package="com.twentydeka.itrash.android"
    android:versionCode="0"
    android:versionName="0" >

    <uses-sdk android:minSdkVersion="8" android:targetSdkVersion="9" />
    <supports-screens android:anyDensity="true"/>

    <!-- The RedLaser SDK requires that these permissions be set -->
    <uses-permission android:name="android.permission.CAMERA" />
    <uses-permission android:name="android.permission.ACCESS_NETWORK_STATE" />
    <uses-permission android:name="android.permission.INTERNET" />
    <uses-permission android:name="android.permission.VIBRATE" />
    <uses-permission android:name="android.permission.WRITE_EXTERNAL_STORAGE" />

    <!-- Additional permission required by GCM and OAuth -->
    <uses-permission android:name="android.permission.GET_ACCOUNTS" />
    <uses-permission android:name="android.permission.WAKE_LOCK" />
    <uses-permission android:name="com.google.android.c2dm.permission.RECEIVE" />
    <uses-permission android:name="android.permission.USE_CREDENTIALS" />

    <permission android:name="com.twentydeka.itrash.android.permission.C2D_MESSAGE"
        android:protectionLevel="signature" />
    <uses-permission android:name="com.twentydeka.itrash.android.permission.C2D_MESSAGE" />

    <application
        android:allowBackup="true"
        android:icon="@drawable/ic_launcher"
        android:label="20deka"
        android:name="com.twentydeka.itrash.android.app.ItrashApplication"
        android:theme="@style/itrash_theme" >

        <!--  activities -->
        <activity
            android:name="com.twentydeka.itrash.android.activity.MainActivity"
            android:label="@string/app_name"
            android:windowSoftInputMode="adjustPan|stateHidden" >
            <intent-filter>
                <action android:name="android.intent.action.MAIN" />

                <category android:name="android.intent.category.LAUNCHER" />
            </intent-filter>

            <intent-filter>
                <action android:name="android.intent.action.SEARCH" />
            </intent-filter>

            <meta-data android:name="android.app.searchable"
                       android:resource="@xml/searchable"/>

        </activity>

        <activity
            android:name="com.twentydeka.itrash.android.activity.product.BarcodeScannerActivity"
            android:label="@string/app_name"
            android:configChanges="keyboardHidden|orientation" >
        </activity>

        <activity android:name="org.acra.CrashReportDialog"
            android:theme="@android:style/Theme.Dialog"
            android:launchMode="singleInstance"
            android:excludeFromRecents="true"
            android:finishOnTaskLaunch="true" >
        </activity>

        <!--  services -->
        <service android:name="com.twentydeka.itrash.android.service.ItrashHttpClientService"
            android:exported="false" >
        </service>

        <service android:name="com.twentydeka.itrash.android.service.ItrashGCMSyncService"
            android:exported="false" >
        </service>

        <service android:name="com.twentydeka.itrash.android.service.ProductSyncService"
                android:exported="false" >
        </service>

        <service android:name="com.twentydeka.itrash.android.service.ConsumerHealthCheckService"
                android:exported="false" >
        </service>

        <!--  providers -->
        <provider android:name="com.twentydeka.itrash.android.contentprovider.ItrashContentProvider"
            android:authorities="com.twentydeka.itrash.android.contentprovider"
            android:exported="false" >
        </provider>

        <!-- receivers -->
        <receiver android:name="com.twentydeka.itrash.android.receiver.GCMSyncBroadcastReceiver"
            android:permission="com.google.android.c2dm.permission.SEND" >
            <intent-filter>
                <action android:name="com.google.android.c2dm.intent.RECEIVE" />
                <category android:name="com.twentydeka.itrash.android" />
            </intent-filter>
        </receiver>

        <receiver android:name="com.twentydeka.itrash.android.receiver.ConsumerHealthCheckBroadcastReceiver" />

    </application>

</manifest>


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These are the first Xbox 360 games you'll be able to play on the Xbox One
By Shawn Knight on November 9, 2015, 17:15 15 comments
Microsoft will soon give Xbox One owners another reason to ditch the 360. Among the many new features of the upcoming Xbox One Experience update will be backward compatibility with select Xbox 360 games.
Mike Ybarra, Director of Program Management for Xbox, revealed on Monday the first 104 backward compatible titles. There are plenty of noteworthy titles on the list including Assassin's Creed II, Borderlands, Dirt 3, Fallout 3, the entire Gears of War collection, Halo: Spartan Attack, Just Cause 2, Mirror's Edge 2, Perfect Dark and Perfect Dark Zero, Sonic the Hedgehog 1-3 and Wolfenstein 3D.
The complete list of backward compatible games can be found over on the Xbox website.
Ybarra added that gamers can expect new backward compatible games to be announced on a regular basis starting in December. Games to look forward to include Bioshock, Bioshock 2, Bioshock Infinite, Call of Duty: Black Ops, Halo Reach, Halo Wars and Skate 3, just to name a few. Fans can also visit the feedback website to vote on existing nominations or to submit their own for consideration.
The update will also introduce fully-customizable Xbox One Avatars and a revamped OneGuide. The former will feature the same visual fidelity improvements (better anti-aliasing, lighting, shading and so on) that gamers on Windows 10 are already enjoying while the new OneGuide brings an enhanced UI and a better live TV experience.
Microsoft will begin the Xbox One Experience update rollout on November 12.
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text:
Residential interior design continues to evolve to include technology as both part of the design and part of the experience. In this Design Showcase, we focus on four brands from across the pond that deliver unique solutions that designers need now to deliver a better home entertainment experience to luxury clientele.
Display Technologies, Meridian, TPI, and Light Walls—all based out of the UK and distributed by Apex Technologies in the United States—offer highly specific design-driven high-performance products that answer problems such as hiding the video display in unusual situations, creating an immersive cinematic experience that uses every part of the room (not just the front wall), and delivering speakers that take home audio to another level in both performance and breathtaking design.
Since 1977, Meridian Audio has been crafting innovative, elegant, high-performance audio solutions. From its flagship DSP (Digital Signal Processing) loudspeakers with integrated amplification to its discreet and powerful range of in-wall loudspeakers, Meridian delivers easy-to-install, high-resolution audio systems for home cinemas and whole home music solutions. The renowned Meridian experience is now available in a variety of environments thanks to its collaborative partnerships with industry leaders like Jaguar, Land Rover, LG Consumer Electronics, and British Airways, ultimately transforming the way people experience sound, wherever they may be.
In the project pictured below, Jason Bellanti, COO and technology integrator at HTA Certified Spire Integrated Systems, had worked with the client in the past. "When she came into the showroom with her interior designer, we had the Meridian DSP7200 speakers on display," says Bellanti. "When the interior designer and the client saw them, she knew she wanted to include them in her room as a functional design element. Then when they listened to them, they were sold." Spire had the black DSP7200s on display, but the client and interior designer opted for a bold, stark white finish to tie the room together.
"Architects love Meridian speakers because they don't require rack space," says Ryan Donaher of Apex. That's because with the Meridian DSP series, the speakers contain internal amplifiers that deliver all the power needed for robust room sound, eliminating the need for separate amplifiers located in a closet somewhere.
Interior designers also love Meridian DSP speakers because they can match other design elements in the room exactly. In one case, the homeowners wanted to match the Roche Bobois cabinet in their living space to the Meridian speakers. Meridian was able to supply the RAL color to the furniture company so that they could customize the cabinet to match the speakers exactly. In another case, the Meridian DSP speakers were custom-painted to match a cappuccino machine!
"Meridian is about design options and performance, even in compromising positions that are less than ideal. In every case, the DSP technology improves performance," says Donaher.
Display Technologies is a British brand specializing in advanced projector screens, mounting brackets and custom installation solutions. Its focus is on the combination of technical performance and design for install, ensuring that all Display Technologies products meet the needs of the homeowner, architect, designer, and installer.
In the image above, you might ask, where is the projector shining onto the screen? It's stashed—incredibly—in a cabinet behind the seating area thanks to a unique mirror mount from Display Technologies, pictured here.
All directions of Display Technologies' Vertical Mirror Mount are now combined to create the DT-VMMv2. With Universal mounting and orientation, this flexible configuration is able to be used with the vast majority of projects, despite space constraints.
TPI Sound
TPI Sound manufactures an exceptional and comprehensive range of professional audio equipment, including loudspeakers, amplifiers, audio processors, and accessories for the studio, high-output, and theater markets.
TPI's team of world-class engineers, led by Giles Smith, designs and builds professional audio equipment which offers "simply the best performance on the planet." TPI's products are manufactured in England to precise military specifications and tolerances, giving you outstanding value and lifelong performance. Their roster of professional users—such as Warner Brothers, Atlantic Records, and Sony Music—who rely on their recording studio systems is a testament to the brand.
The TPI Professional Cinema line delivers the opportunity to bring reference-quality cinema playback into the home environment.
TPI has several key technologies that allow their speakers to produce very high SPL (sound pressure levels) with Soft Dome Tweeters, achieving professional power output while maintaining high-fidelity musicality.
There is also a wide range of 4" deep loudspeaker models, all of which achieve the required performance criteria for Dolby Atmos Professional playback. "This is a very big deal as most other professional speakers are designed for commercial spaces, not homes. TPI brings Dolby Professional-compliant product to the residential market, with a 4" deep custom install solution," says Donaher.
If you or your client want a world-class luxury home theater, with professional performance credentials, TPI is an incredible brand worth considering.
Light Walls
Light Walls is the go-to dynamic LED lighting partner and home of innovative immersive and dynamic LED-based lighting for creative residential and commercial applications. With ready-made or custom solutions, Light Walls helps you deliver projects from modest linear LED Dynamic Lighting systems, to immersive video systems, artistic creations, and large-scale commercial installation. Light Walls has a wide portfolio of products matched with design expertise, creative vision, and manufacturing capabilities to enable you to realize unique and exceptional experiences.
From a design perspective, there is no end to how creative a designer, architect, or integrator can get with Light Walls technology. "Light Walls can bring nature in and create a view of the forest. We even have an integrator who outfitted the Omaha Natural Gas Company using Light Walls to create images of faux pipes in the ceiling that look as though gas is running through fake pipes," says Ryan Donaher of Apex Technologies. "The exciting component is LED lighting technology that adds motion and movement into light that has never been done before. Any creative person will jump all over this." From an integration perspective, it's all done with minimal rack space and is amazingly inexpensive compared to direct view video walls.
So whether it's jaw-dropping audio performance, breathtaking sculptural speakers, unique video display solutions for tricky rooms, or new lighting technology, Apex delivers with its portfolio of products.
"One of the things that sets Apex apart is we offer design services. Our partners can call us if they have a project, and we help them specify product working directly with these brands," says Donaher. "We abide by all standards from Dolby, CEDIA, and others to make sure that our technology integrators are delivering exactly what the homeowner wants. We want to know that our products are being installed with an awesome team behind them, and not a guy working out of a van." To that point, almost every one of Apex's dealers are HTA Certified!
Find an integrator near you here, and reach out to the team at Apex by clicking the phone or email icons to the right (or below if you're on a mobile device).
The Moving Canvas - 21st Century Digital Art Arrives In the Home
Why a Smart Home Needs a Strong Wiring Backbone
Low-Voltage Lighting by Colorbeam Brings Beverly Hills Estate to Life
Home Office 2.0 - Top Tips for a Great Home Office


text:
RubyMaybe
Translating Korean Novels
Volcanic Age
Lucia Chapter 31 [part 1]
June 22, 2018 April 17, 2019 / Miss Ruby
PREVIOUS |INDEX| NEXT
< — Damian — > (2)
TN: Enjoy~~
A jet-black carriage hurried across the streets of Roam.
Taking a closer look at the little black wooden carriage, one could find the motif of a black lion drawn on it.
The black carriage was so fascinating that people stopped what they were doing to look at it when it first appeared.
The black wood used as the main material of the carriage was as strong as steel, and is said to have been used by the military in the past.
And because many people withered to death from the sickness gotten from the natural habitat of the black wood, the price of the black wood had now surpassed that of gold.
Hugo had made this carriage of black wood for the safety of his wife, so Lucia often went out in a carriage that was fit for a King.
At this point, people already knew who was inside whenever the black carriage passed by.
Most people would never get to see the faces of the ones inside such a carriage in their lifetime because the ones inside were in such a high position.
And so, when the carriage appeared, they just watched it till it was out of their sight.
When the carriage crossed the bridge and entered the gates, the sound of a horn was heard. The black horse carriage carrying Lucia continued to run and stopped in front of the deepest inner tower in the Roam Castle.
The servants were all outside to greet the Lady. When Lucia returned from her horse riding, just as usual, she took a bath then sat in the receiving room, drinking the fragrant tea that Jerome served.
"Did you enjoy your outing, Milady?" (Jerome)
"I enjoyed it. Emily is a really kind child, she followed my clumsy instructions very well."
Her favorite horse, Emily was a nice and well-trained mare that Hugo had given to her.
Lucia didn't really know much about horses but when she looked at its glossy appearance she could guess it was a good horse.
Whenever Lucia heard compliments about its loveliness, she just shrugged her shoulders.
"Who said such a thing? Even if you had 10 horses, you can't replace Emily. She is a very expensive horse."
"Yes. It seems that way."
As it was not polite to discuss the price of his master's gift, Jerome did not say the details. Lucia didn't ask either, but she was thankful that Hugo thought about her and gave her precious horse as a gift.
'I miss him…'
"When is he coming back?"
"Yes? Ah… I don't know exactly, but it can be long. I think it will be about a month."
"A month…? What exactly is going on? I know it's work in the fief…"
Before, she was not interested in what he was doing, but now she wanted to know more about it.
"Some parts are about the fief, others are not. It is something Master goes to do annually."
Jerome tried to emphasize that his master's suddenly departure was only for work reasons and definitely not for a private reason. He did not know about the dramatic reconciliation of the couple.
"Milady already knows that the northern border is close to the barbarians. They is no central tribe so from time to time, they cross the border to plunder. Master subdues them once a year to keep them in check."
"Then, every year, he leaves at this time of the year?"
"This year is earlier than other years. He usually leaves early in the winter. I heard that an order to dispatch arrived. It seems that the barbarians haven't been payed much attention to after the war, so their plundering became more frequent."
"It must be hard for the Northern people who continuously get anxious because of this."
"If they do not live close to the border, I do not think they would be anxious. Things are quite different when you looks at it from afar."
Lucia nodded her head, sipping a little tea then suddenly exclaimed.
"Oh my goodness! How could I forget? Isn't today his birthday Jerome?"
The birthday date that Jerome had reminded her of before, was exactly today. She had kept it in her mind but had forgotten about it because they were fighting.
"I should have told him before he left. It's his birthday but he won't receive any congratulation, he'll just be fighting barbarians."
She felt so sad for him that her heart hurt.
"Mmm… Milady, Master had never taken care of his birthday separately."
"I expected that. Who would take care of his birthday like that? Only the people around him can."
"That…he doesn't like being reminded of it."
"I don't really know much. However I often get the feeling that not only about his birthday, Master also dislikes being reminded of his childhood."
Jerome never spoke of anything he was unsure of or carelessly speak his mind but because she looked very sad, he gave a sincere reply.
'So he doesn't have any memories he wants to remember from his childhood…'
That was a sad thing.
Lucia lived a difficult life and there was a moment in her life where her memory was perfect. She was happy until she was twelve years old. The times when she lived with her mother as a child were happy times.
The story of the Duke's tragedy in the west tower once again rose to the forefront of Jerome's mind. It was a terrible thing at the time and he shouldn't think of it nor speak of it so he tried to forget it.
However the thought always arose whenever he laid eyes on the west tower, and as time went on, he grew more interested in the background story than the murder itself.
The dead Duke had abandoned one of his sons to stop misfortune and left him to his own fate. As he did something no parent should ever do, he brought it upon himself.
"Jerome, you said you've never met the late Duke before, right?"
"Yes. I have served master ever since he became a knight."
"This may just be my prejudice but I think he was a very heartless man."
Jerome hesitated for a moment before speaking.
"From the little fragments that I have gathered, I don't think my thoughts differ very much from Milady's."
Lucia, Translations
Fun to Translate
← Lucia Chapter 30 [part 2]
Lucia Chapter 31 [part 2] →
10 thoughts on "Lucia Chapter 31 [part 1]"
Mushroom Head
Thank you 😗 Damian oh Damian, where are you ?😂
grifttail
F_J
Wait. Did they make up?? I'm kinda lost? And when is the little boy coming back? Lucia needs some company~~
Miss Ruby
Yes. They made up in 29.2
Demian won't you show yourself??
shirotan
Funny how we don't care about Hugo now that he's for a month XD
We just care about Damien and how he's going to just loooooove Lucia XD
The next 10? chapters are about Damien? It's too hard to stock up the chapters to read T_T
Thanks for the translations!!!!
Whaa, I care about Hugo…. Damian just has more of my love atm 😇❤
fan63
I care for Hugo as well. He needs Lucia to stay alive in his spirit.
Zuridah Hani
Thank youuu
Get releases in advance!
Lucia Chapter 116 [part 2]
There will be 3 releases per week. For information on advance chapters, click here
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Exclusive property with excellent sea views. A large lawned garden lies before the house and at the rear is the pool area.
The main feature of the house are the large windows, aluminum double glazed, not to lose any of the rooms in the wonderful views. The house consists of three bedrooms and four bathrooms. Both bedrooms are suites plus a third bedroom with external bathroom located on the second floor. Radiant heating offers great comfort throughout. There is pre-installation for the elevator.
Upstairs a loft fully equipped as an office and gym. On the lower floor, basement, is the two-car garage.


Since its renovation in 2002, Brick-5 has quickly become one of the most exciting alternative venues on Vienna's cultural scene. A building with a rich and varied history, Brick-5 is now home to a wide range of concert, theater, visual art and dance events. Entrance to the performance space is via the Turnhalle, a trendy ground floor bar and restaurant which is open before, during and after performances. The 200m² lofted performance space itself provides wonderful acoustics and an inviting atmosphere ideal for Mosaïque programs.
Don't let the address frighten you. BRICK-5 is a stone's throw from the Gürtel, MUCH closer to the center than you might think!


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The new version of the exam is still over a year away. ARE 5.0 is not set to launch until late 2016, well over a year from now. Anyone that is currentlytesting under version 4.0, or anyone who is thinking that they're just about ready to get started, still has plenty of time to finish testing beforehand. There's absolutely no need to feel rushed just because a new version of the test is on its way.
Even after ARE 5.0 launches, ARE 4.0 will still be around for another 18 months. ARE 4.0 won't be retired until June 2018. This means that both versions of the exam will be available for that period of time, which NCARB is calling "dual delivery." Again, plenty of time for you Four-Oh-ers to finish testing… but one has to wonder if Prometric's testing centers might find themselves just a little more crowded with two versions of the ARE being administered… That might be more than enough incentive to get the test out of the way.
Content for ARE 5.0 is still being developed. At this time, we know that there will be six divisions instead of seven, that content will be more equally spread out between those six (translation: there's no dedicated structures exam, but you can expect to find structural-related content on all six divisions), and that the much-maligned graphic vignettes are being retired in favor of more sophisticated graphic-based problems that might require the user to, say, drag and drop a piece of through-wall flashing to the correct area of a wall section. Other item types include a case study that candidates will need to read, then answer a series of related questions afterward. Needless to say, this test will be a completely different animal than 4.0. Exam guides and other review materials will begin to be made available until early 2016, and I'm sure that NCARB will continue to keep us updated along the way.
Eligibility will be key. So, yes, ARE 4.0 will still be around until 2018, and, yes, that Credit Model is brilliant. That doesn't mean you should rest on your laurels. Once ARE 5.0 launches, candidates will no longer be able to gain eligibility to test under ARE 4.0. What does that mean? It means that if you're thinking that you're ready to begin testing in the next year, get your authorization to test as soon as possible! Rather than limiting yourself to only version 5.0, give yourself as many tactical advantages as possible over the exam by allowing yourself the ability to test in both versions.
The most important thing to remember is that, even though ARE 5.0 is on the horizon, ARE 4.0 is still the current version of the exam. To that end, NCARB has continued to develop and launch even more resources dedicated to helping candidates complete ARE 4.0, including their excellent Google+Community, a series of YouTube videos for each division of the test, and an updated batch of Exam Guides, with new multiple choice sample questions. And don't forget – the AIA is here to help! Both the Pittsburgh and Philadelphia chapters offer monthly review sessions, and most chapters maintain a lending library of study resources. After all, the best way to avoid any potential confusion about ARE 5.0… is to just knuckle down and get 4.0 out of the way! Contact your local AIA Chapter office or AIA Pennsylvania for more information about the resources that are available in your area, and feel free to drop me a line if you have questions about IDP, ARE, or anything in between.
A version of this article originally appeared in AIA Pittsburgh's Columns.


text:
Statistical Biology Team
CIRB Collège de France
Data/codes
O. Rivoire. Parsimonious evolutionary scenario for the origin of allostery and coevolution patterns in proteins. arXiv:1812.01524.
A. S. Zadorin. Allostery and conformational changes upon binding as generic features of proteins: a high-dimension geometrical approach. arXiv:1905.02815.
A. F. Schober, A. D. Mathis, C. Ingle, J. O. Park, L. Chen, J. D. Rabinowitz, I. Junier, O. Rivoire, K. A. Reynolds (2019). A two-enzyme adaptive unit in bacterial folate metabolism. Cell Reports 27 : 3359-3370. [SI]
I. Junier, P. Frémont, O. Rivoire (2018). Universal and idiosyncratic characteristic lengths in bacterial genomes. Phys. Biol. 15 : 035001. [SI]
A. Mayer, T. Mora, O. Rivoire, A. M. Walczak (2017). Transitions in optimal adaptive strategies for populations in fluctuating environments. Phys. Rev. E 96 : 032412.
A. Mayer, T. Mora, O. Rivoire, A. M. Walczak (2016). Diversity of immune strategies explained by adaptation to pathogen statistics. PNAS 113 : 8630–8635. [SI]
O. Rivoire, K. Reynolds, R. Ranganathan (2016). Evolution-based functional decomposition of proteins. PLoS Comput Biol 12 : e1004817. [SI]
I. Junier, O. Rivoire (2016). Conserved units of co-expression in bacterial genomes: an evolutionary insight. PLoS ONE 11 : e0155740. [SI]
S. Boyer, D. Biswas, A. K. Soshee, N. Scaramozzino, C. Nizak, O. Rivoire (2016). Hierarchy and extremes in selections from pools of randomized proteins. PNAS 113 : 3482-3487. [SI]
O. Rivoire (2016). Informations in models of evolutionary dynamics. J. Stat. Phys. 162 : 1324–1352.
M. Hemery, O. Rivoire (2015). Evolution of sparsity and modularity in a model of protein allostery. Phys. Rev. E 91 : 042704.
O. Rivoire, S. Leibler (2014). A model for the generation and transmission of variations in evolution. PNAS 111 : E1940-E1949. [SI]
O. Rivoire (2013). Elements of coevolution in biological sequences. Phys. Rev. Lett. 110 : 178102. [SI]
O. Rivoire, S. Leibler (2011). The value of information for populations in varying environments. J. Stat. Phys. 142 : 1124-66.
R. Smock, O. Rivoire, W. Russ, J. Swain, S. Leibler, R. Ranganathan, L. Gierasch (2010). An interdomain sector mediating allostery in HSP70 molecular chaperone. Mol. Syst. Biol. 6 : 414. [SI]
N. Halabi, O. Rivoire, S. Leibler, R. Ranganathan (2009). Protein sectors: evolutionary units of three-dimensional structures. Cell 138 : 774-86. [SI]
Olivier Rivoire's complete list of publications
Clément Nizak's complete list of publications