Team:EPF-Lausanne/Notebook/August2011

From 2011.igem.org

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(Wednesday, 24 August 2011)
 
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Vincent and Alessandro did a colony PCR with Taq polymerase using the K1 transformations done on Thursday. The C5 transformations yielded a small number of colonies. The gel result is below. The bright bands correspond to K1-T7-const-RFP, K1-T7-lac-FRP, K1-T7-lac2-RFP.  
Vincent and Alessandro did a colony PCR with Taq polymerase using the K1 transformations done on Thursday. The C5 transformations yielded a small number of colonies. The gel result is below. The bright bands correspond to K1-T7-const-RFP, K1-T7-lac-FRP, K1-T7-lac2-RFP.  
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[[File:t7_lac_lys_RFP_gel.tif]]
 
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[[File:t7_lac_lys_RFP_gel.jpg|600px]]
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After noticing the poor results for the lysis, Alessandro and Vincent realized that they had set the extension PCR extension time to 1 minute rather than the 2 that are needed for the lysis cassette (which has a length of 2 kb).
Lilia compared the received sequences of the TetR variants to the expected.
Lilia compared the received sequences of the TetR variants to the expected.
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# PK39 variant was already made: [https://static.igem.org/mediawiki/2011/5/57/EPFL2011_muTetR_PK39_BP3col1.pdf see PK39]   
# PK39 variant was already made: [https://static.igem.org/mediawiki/2011/5/57/EPFL2011_muTetR_PK39_BP3col1.pdf see PK39]   
# YF42 variants:  [https://static.igem.org/mediawiki/2011/0/0b/YF42_BP4col1.pdf "YF42"]  [https://static.igem.org/mediawiki/2011/3/33/YF42KE108_BP4col6_.pdf "YF42KE108]  
# YF42 variants:  [https://static.igem.org/mediawiki/2011/0/0b/YF42_BP4col1.pdf "YF42"]  [https://static.igem.org/mediawiki/2011/3/33/YF42KE108_BP4col6_.pdf "YF42KE108]  
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# PQ39YM42 variants:  [https://static.igem.org/mediawiki/2011/6/68/PQ39YM42LP52_BP5col6.pdf "PQ39YM42LP52"]  [https://static.igem.org/mediawiki/2011/6/6c/PQ39YM42_BP5col4_insertion_deletion.pdf "PQ39YM42_ins_del"]  
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# PQ39YM42 variants:  [https://static.igem.org/mediawiki/2011/6/68/PQ39YM42LP52_BP5col6.pdf "PQ39YM42LP52"]  [https://static.igem.org/mediawiki/2011/6/6c/PQ39YM42_BP5col4_insertion_deletion.pdf "PQ39YM42_ins_del"]
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== Saturday, 20 August ==
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As part of Friday's Colony PCR, Alessandro and Vincent had made liquid cultures and made separate, dedicated plates for each colony that was used. Since the Colony PCR had successfully amplified the K1-T7-RFP plasmids, Vincent brought out those liquid cultures, made glycerol stocks of them, mini-preppred them with the new mini-prep kit, and recorded the resulting DNA concentrations. With these DNA tubes, he went ahead and made a transformation of the three plasmids into BL21 cells (which Alessandro had made throughout the past week). Vincent plated these transformed cells on kanamycin plates and incubated them overnight.
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Since the previous day's Colony PCR had partly failed due to an extension time for lysis that was too short (1 minute rather than 2 minutes), Vincent made a Colony PCR for K1-T7-Lysis and a Colony PCR for C5-T7-RFP and C5-T7-Lysis. Perhaps because it was almost 9 in the evening, Vincent did not realize that he had successfully amplified both the K1 and the C5 T7 lysis, showing that the Gibson Assembly had in fact worked as expected. He saw the primer dimers at the bottom of the gel and did not think to even look for faint bands at the 2 kb level where the lysis piece would appear. So the Gibson did in fact work, but Vincent only found this out retrospectively on Tuesday of the following week (this wiki post is written with some tardiness).
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[[File:vince_gel_c5rfp.jpg|600px]]
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== Sunday, 21 August ==
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Vincent took out the plates from the BL21 transformation. The results were spectacular: thousands of colonies. He then picked one colony from each plate to use for liquid cultures. He did not forget the antibiotic (as he usually does). The idea was to have liquid cultures ready for an IPTG platereader experiment on Monday.
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Vincent also ran a miniprep on the C5-T7-RFP cultures, but got a very low yield of DNA (9-10 ng/uL).
== Monday, 22 August 2011 ==
== Monday, 22 August 2011 ==
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She also made negative controls, where only the backbone has been mixed with water, and transformed everything.
She also made negative controls, where only the backbone has been mixed with water, and transformed everything.
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Vincent ran a platereader experiment using the liquid cultures of T7-RFP plasmids. Results should be appearing shortly.
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Vincent received all 18 of the primers he needed to make the T7 promoter variants. Here is the list of these primers (this indexing will be used in the gel pictures):
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1. T7 const
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2. T7 014
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3. T7 030
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4. T7 054
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5. T7 080
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6. T7 111
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7. T7 const (-7-12)
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8. T7 const (-3-1/+1+3)
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9. T7 const (-10)
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10. T7 lac2
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11. T7 lac2 014
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12. T7 lac2 030
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13. T7 lac2 054
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14. T7 lac2 080
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15. T7 lac2 111
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16. T7 lac2 (-7-12)
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17. T7 lac2 (-3-1/+1+3)
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18. T7 lac2 (-10)
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Vincent made 50 uM and 500 nM dilutions of the primers.
== Tuesday, 23 August 2011 ==
== Tuesday, 23 August 2011 ==
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The Gibson-transformed cells yielded a lot of mutants, with significantly more on the Gibson plates than on the negative controls ones. One strange thing with the J61002 Plac-RFP: the negative control  cells were weakly red fluorescent, while for the Gibson assembly some of the cells were expressing RFP. We'll see the results of the colony PCR tomorrow, but it's possible that the cells are autofluorescent...
The Gibson-transformed cells yielded a lot of mutants, with significantly more on the Gibson plates than on the negative controls ones. One strange thing with the J61002 Plac-RFP: the negative control  cells were weakly red fluorescent, while for the Gibson assembly some of the cells were expressing RFP. We'll see the results of the colony PCR tomorrow, but it's possible that the cells are autofluorescent...
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Vincent made an extension PCR for both the RFP and Lysis T7 promoter variants (36 samples). The extension PCR had an elongation time of 1 min. or RFP and 2 min. for Lysis. The protocol is avaible in the protocol section.
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[[File:Rfp_t7_variants.jpg|600px]]
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[[File:Lys_t7_variants.jpg|600px]]
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Clearly the RFP T7 promoter variants went well, but the T7 Lysis promoter variants ... not so much. Vincent made liquid cultures of all the T7 lysis plasmids (both C5 and K1) and put them in the incubator. He also pelleted all the C5 RFP liquid cultures and the K1-Lys cultures that he had made on Monday (6 colonies per promoter) thinking that he had not yet achieved a full T7-lysis. The pellets are being kept in a special box called "Pellets".
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Lilia made linear templates out of the mutants plasmids, here the T7 promoter was added to be able to use it for in vitro translation/transcription with the TNT T7 ITT wheat germ extract kit.
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[[File:EPFL2011_muTetRs_linear_template_PCRresults.png|350px]]
== Wednesday, 24 August 2011 ==
== Wednesday, 24 August 2011 ==
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[[File:EPFL_Igem_2408_psbColPCR.jpg|thumb|300px|Colony PCR on pSB3K1 TetR-LacI assembly]]
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[[File:EPFL_Igem_2408_j6RFPcloPCR.jpg|thumb|300px|Colony PCR on J61002 Plac-RFP assembly]]
Nadine ran the colony PCRs on a gel and got strange results.  
Nadine ran the colony PCRs on a gel and got strange results.  
* For J61002 Plac-RFP, the negative control cells are definitely red - although they should not contain RFP -  and the colony PCR that includes a primer on RFP is positive. The colonies 8-10 were still white on the plate, but here again the colony PCR is positive. Perhaps this comes from the template plasmid used for the PCR (J61002 Ptet_EFP); since the miniprep failed we have to wait before sending the samples for sequencing.
* For J61002 Plac-RFP, the negative control cells are definitely red - although they should not contain RFP -  and the colony PCR that includes a primer on RFP is positive. The colonies 8-10 were still white on the plate, but here again the colony PCR is positive. Perhaps this comes from the template plasmid used for the PCR (J61002 Ptet_EFP); since the miniprep failed we have to wait before sending the samples for sequencing.
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* One for amplifying LacI and put it into J61002 Plac-RFP or Plac-lysis, once we'll have them.
* One for amplifying LacI and put it into J61002 Plac-RFP or Plac-lysis, once we'll have them.
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{{:Team:EPF-Lausanne/Templates/Footer}}
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[[File:EPFL2011_TetR_variants_list_24.08.11.jpg|700px]]
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Vincent mini-prepped the liquid cultures of the original K1-T7-Lys plasmids that had been (unknowingly) successful on Saturday. He then transformed these into BL21 cells and plated them. The C5-lys plasmid liquid cultures did not grow because Vincent had accidentally used gentamycin instead of chloramphenicol in his liquid cultures. He re-did the C5 cultures.
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Vincent also ran a new set of 18 T7-Lys promoter variants using Extension PCR. Because we were running out of Hifi+ enzyme, we used only half of the enzyme requirement (9 uL instead of 18).
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[[File:T7lysis_variants.jpg|600px]]
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Apparently, 14 through 17 were amplified correctly.
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Lilia made a PCR with the primers containing biobrick extentions (prefix and sufix) on the linear templates of the muTetRs.
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[[File:EPFL2011_muTetRs_1st_biobrick_PCR_on_linear_template.png|350px]]
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== Thursday, 25 August 2011 ==
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The sequencing results of the pSB3K1 TetR-LacI plasmid came. It seems that LacI got insterted correctly, but that TetR got lost... We'll try to send this plasmid again for sequencing with another primer to be sure. Also, a new Gibson assembly has been done.
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For the J61002 Plac-RFP plasmid, Nadine  forgot the cell cultures on the bench so the sequencing will have to wait over the week-end. Here again, a new Gibson assembly has been done, along with J61002-lysis.
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Vincent made liquid cultures from the successful transformation of the K1-T7-Lysis into BL21 cells. Nadine was kind enough to purify all 18 of Vincent's T7-RFP variants, as well as the 4 T7-Lysis variants. In the same spirit, the C5-T7-Lysis plasmids were miniprepped and then transformed into the BL21 cells and plated.
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In order to continue with the production of T7 promoter variants, Vincent had to make more gene-specific Lysis (i.e. T4 Lysis with RBS upstream). For this, Vincent used the new Hifi+ enzyme that had been ordered the previous day. Since we were also running out of PCR primer mix, Vincent went ahead and made some more. The gel result for the T4 Lysis cassette is found below.
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[[File:Genespec_lys.jpg|600px]]
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== Friday, 26 August 2011 ==
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Vincent ran a plate-reader experiment for the K1-T7-lysis BL21 cells. The first three rows received various doses of IPTG before the experiment got under way. The next three rows (const, lac, lac2) received no IPTG for the first 2 hours of incubation but then were given the same IPTG doses as the first three rows. Data analysis will take place tomorrow.
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Vincent also made liquid cultures of the C5-T7-lysis BL21 cells. After consultation with Henrike, we have decided to drop the C5 backbone since it does not really add to the work we are doing and only adds more PCRs and cultures.
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Vincent made a Gibson assembly of the 18 T7-RFP promoter variants and the 3 T7-Lysis variants into the K1 backbone. The DNA will be transformed on Saturday.
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[[File:EPFL_Igem_2608_j6RFPcol24.jpg|thumb|200px]]
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[[File:EPFL_Igem_2608_pcr13.jpg|thumb|200px]]
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Nadine made colony PCRs for yesterday's Gibson assemblies: J61002 Plac-RFP and pSB3K1 TetR-LacI; she also made PCRs for pSB3K1 TetR-LacI that had been sent for sequencing on wednesday. Unfortunately, the plate with J61002 Plac-lysis yielded no colonies...
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Details of the PCRs:
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* pSB3K1 TetR-LacI:
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** amplify LacI with sequencing primers
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** amplify TetR with sequencing primers
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* J61002 Plac-RFP:
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** amplify RFP with colony PCR primers
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** amplify RFP with Gibson primers
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With the sequencing primers, one of them binding in the middle of RFP, all 6 colony tested gave positive results. Strangely, there seems to be 2 bands with a slight difference in size. With the Gibson primers that contain Plac, colonies 1 and 3 have a brighter band. Either they are the only positive ones, or they just have a bigger amount of DNA (as also seen on the other gel).
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For pSB3K1, both PCRs failed. Since it's very unlikely that we have a plasmid that lost TetR, there must have been a problem in the PCR; it will be repeated.
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[[File:EPFL_Igem_2608_J6RFP_dig.jpg|thumb|200px]]
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Nadine also digested the (supposed) J61002 Plac-RFP plasmids from Tuesday's Gibson assembly: colony 6 from Gibson plate that was red, colony 9 from Gibson plate that was white and colony 12 from negative control plate that was also red. Pst1 has a restriction site in the original J61002 backbone, therefore it should cut independently of RFP insertion. The results indicate that only colony 9 was digested by the enzyme, therefore it's likely that the red colonies contain an unknown plasmid that has RFP... Since colony 9 was positive for RFP colony PCR, it can be our expected plasmid that has little expression of RFP.
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== Saturday, 27 August 2011 ==
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[[File:Igem_2708_repcr.jpg|thumb|300px]]
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Nadine did a second colony PCR on the pSB3K1 TetR-LacI colonies. This time, the LacI PCR worked for all samples (fragment should be about 750 bps), but the TetR PCR failed again (although these primers already worked in the past). One colony will be sent for sequencing on Monday.
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Vincent first made Nanodrop measurements of his Gibson assmebly DNA (post PCR purification from Friday night). The concentrations were abysmally low, which made it likely that the transformations would be unsuccessful. Nevertheless,
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the Gibson Assemblies of the K1-T7-RFP variants (1-18) and the three K1-T7-Lysis variants (14, 16, 17) were transformed into DH5 alpha cells and plated.
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== Sunday, 28 August 2011 ==
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What a surprise on Sunday afternoon to find that all the transformations -- both RFP and Lysis -- had made dozens of colonies each! With all these beautiful colonies just begging for a Colony PCR, Vincent could not resist and did a colony PCR as well as liquid cultures (and a large plate) for all 21 plates.
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[[File:T7lysisgibson_check.jpg|600px]]
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[[File:T7rfpvariants_gibsoncheck.jpg|600px]]
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In an attempt to finish off the remaining stubborn T7-Lysis variants, Vincent used the gene-specific T4-Lysis product from Friday to start an Extension Touchdown PCR with a Final PCR -- all of which were successful!
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[[File:Remaining_t7lysis_variants_gel.jpg|600px]]
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Vincent also made liquid cultures of BL21 K1-T7-Lysis (non-variant) for const, lac, and lac2 for a Berkeley-style experiment. The platereader showed a behavior that is atypical of BL21 cells.
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Vincent also made a miniprep of Nadine's 3 colonies (J6 1, 4; pSB3K1 9) for sequencing on Monday.
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== Monday, 29 August 2011 ==
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Some brainstorming with Henrike on the topic of the failed IPTG platereader experiment made it clear to Vincent that he had been using the wrong colony plate to make his T7-lysis liquid cultures. Having cast light on a potentially crucial mistake, we then thought it would be a good idea to do another platereader experiment, this time with the BL21 cells and not DH5 alpha.
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Before getting to work on those new liquid cultures, Vincent made the premixed tubes for Microsynth sequencing of Nadine's colonies. Results should be coming in on Tuesday by email. Lilia and Vincent then had an interview with Stéphane of the BioSafety committee at the EPFL. His comments and insights will soon be finding their way to the wiki safety page.
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Henrike, Clara, and Matt helped Vincent mini-prep the 16 T7-RFP and T7-Lysis variants and measure the DNA concentration. They transformed and plated these and the T7-const-LYS, T7-lac-LYS, and T7-lac2-LYS into BL21 cells and also transformed and plated the pSB3K1 plasmid from Gibson assembly (negative control) into DH5 alpha. Vincent had not realized that the plasmid he Gibson-assembled did not have extensions and that there was no need to put this into DH5 alpha. Since Vincent made pSB3K1 extended for the next day's Gibson assemblies (of the remaining RFP and Lysis variants), this only put us behind by one day.
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[[File:Psb3k1_forgibson.jpg|600px]]
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== Tuesday, 30 August 2011 ==
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Though they are exactly one week late, here are the fluorescence and optical density results for the RFP IPTG induction platereader experiment we ran last Monday.
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[[File:T7const_rfp_iptgtest_0822_OD.jpg|300px]]
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[[File:T7lac_rfp_iptgtest_0822_OD.jpg|300px]]
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[[File:T7lac2_rfp_iptgtest_0822_OD.jpg|300px]]
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[[File:T7const_rfp_iptgtest_0822_RF.jpg|300px]]
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[[File:T7lac_rfp_iptgtest_0822_RF.jpg|300px]]
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[[File:T7lac2_rfp_iptgtest_0822_RF.jpg|300px]]
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Vincent purified his PCR products ahead of the Wednesday Gibson assembly. He also made liquid cultures of the 1-5, 7, 9, 10, 11, 12, 15, and 18 T7-RFP variants and the T7-const/lac/lac2 Lysis promoters and the 3 variants (14, 16, 17). These are for a lysis and RFP experiment with IPTG planned for Wednesday.
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Vincent received the sequences for Nadine's 3 colonies (J6 1 & 4 Plac-RFP, K1-TetR-LacI 9) but did not have time to analyze them.
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== Wednesday, August 31 2011 ==
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Vincent made new Gibson master mixes using TAQ ligase (there are now 12 tubes that can do 2 Gibsons each).
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Vincent ran a plateread er experiment with thoe three T7-lysis constructs (const / lac / lac2) and the various T7-RFP variants. It looks as though lysis was induced in at least one T7 system. Vincent made more liquid cultures of the T7-lysis systems and their variants for another platereader experiment on Thursday.
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Vdog also made a Gibson of J6 Plac-Lys and transformed it into BL21 cells (plated all 300 uL). He made another Gibson attempt at the elusive K1-Tet-Lac and transformed that into the DH5 alpha cells (plated 150 uL). He did not forget that one of the plates had ampicillin resistance and the other had kanamycin.
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Vincent also made a colony PCR of the remaining 5 (out of the 18) RFP variants in DH5 alpha. He chose different colonies from the plates to see if those would fare better.
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He also tried to clone the version of the psB3K1 backbone that does not have TetR (we think it's the one that's labelled 1:10 on the eppendorf top but we're not sure). He used the K1-extensino primers. Hopefully, these will be good for Gibsoning the remaining T7-Lysis variants into K1. It will also be used to make the negative control (co-transformed with RFP, eventually).

Latest revision as of 09:04, 21 September 2011