Team:EPF-Lausanne/Notebook/August2011

From 2011.igem.org

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(Monday, 29 August 2011)
(Wednesday, 24 August 2011)
 
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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".
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:T7lysis_variants.jpg|600px]]
[[File:T7lysis_variants.jpg|600px]]
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Apparently, 14 through 17 were amplified correctly.  
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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]]
== Thursday, 25 August 2011 ==
== Thursday, 25 August 2011 ==
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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.
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]]
== Tuesday, 30 August 2011 ==
== 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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{{:Team:EPF-Lausanne/Templates/Footer}}
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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