Team:EPF-Lausanne/Notebook/September2011

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After taking a closer look at the plate from the platereader, it seems that none of the T7 variants did any lysing. This realization made it urgent that we discover whether or not the full T4-lysis cassette was really in these Gibson-assembled plasmids. The samples that had been sent in for sequencing had primers that seemed to amplify the backbone, instead of the lysis cassette. While this does not necessarily mean that the lysis is not there, it does suggest that all the PCRS and gels used to verify the presence of lysis were only showing the existence of the backbone.  
After taking a closer look at the plate from the platereader, it seems that none of the T7 variants did any lysing. This realization made it urgent that we discover whether or not the full T4-lysis cassette was really in these Gibson-assembled plasmids. The samples that had been sent in for sequencing had primers that seemed to amplify the backbone, instead of the lysis cassette. While this does not necessarily mean that the lysis is not there, it does suggest that all the PCRS and gels used to verify the presence of lysis were only showing the existence of the backbone.  
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Alina ran a PCR with different primers from Douglas' first attempts at putting together the 2700 bp cassette that amplify select regions of the cassette.  
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Alina ran a PCR with different primers from Douglas' first attempts at putting together the 2700 bp cassette that amplify select regions of the cassette.
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Lilia tested the expression of the TetR mutants with the T7 promoter. The expression mix contained the Green Lysine and the gel fluorescence was imaged and is shown on the next picture.
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[[File:EPFL2011_SDS-PAGE_ITT_expression_check_muTetRs_T7.png|400px]]
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The BP2col2 does not express, what explains the failure of the protein pool down at the MITOMI made on Wednesday.
== Friday, September 2 2011 ==
== Friday, September 2 2011 ==
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[[File:EPFL_Igem_1309_backbone.jpg|thumb]]
[[File:EPFL_Igem_1309_backbone.jpg|thumb]]
Nadine re-did the PCR to amplify pSB3K1 backbone, this time using the right template DNA and it worked! She had strange results on the platereader experiment again, but it's because she diluted cells in water instead of LB medium. The experiment will be re-ran a 3rd time!
Nadine re-did the PCR to amplify pSB3K1 backbone, this time using the right template DNA and it worked! She had strange results on the platereader experiment again, but it's because she diluted cells in water instead of LB medium. The experiment will be re-ran a 3rd time!
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About the T7 biobricks, the sequencing results show that we already have T7 const (1) and T7 Lac2 030 (12). The other most interesting promoter variants are: T7 030 (3), T7 054 (4), T7 111 (6), T7 lac2 054 (13) and T7 lac2 111 (15). We will focus on them and try to build biobrick formats before the deadline.
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== Wednesday, 14 September 2011 ==
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Nadine tried to purify the PCR products of the TetR mutants, but lost them during the process... So she re-ran the PCR and used it directly do do Gibson assemblies that she later transformed into DH5alpha cells.
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[[File:EPFL_Igem_tetrmutants.jpg|200px]]
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She looked at the plates containing the biobrick variants and made colony PCRs on five colonies of variats 3,4 and 15. The resuklts show that we have 2 good colonies for 3 and one for 5.
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[[File:EPFL_Igem_1408_t7.jpg|200px]]
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Finally, the Plac promoter being correctly inserted into J61002 Plac-RFP plasmid, Nadine also ran a PCR to add biobrick extensions to the promoter.
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[[File:EPFL_Igem_1409_PlacBB.jpg|200px]]
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Vdog ran a ten-hour experiment with the help of Henrike and Matt. We made two 100 mL batches LB with Kan and Amp in 1:1000 dilutions. We then took the two sets of four tubes of 5 mL liquid cultures (of C2-Lys-Ptet-RFP & C11-Lys-Ptet-RFP) and spun them down, threw away the supernatant, resuspended them in 5 mL of PBS buffer, spun them down again, resuspended once more in 5 mL of PBS buffer, and then spun them down again before resuspending one last time in 1 mL of PBS buffer. The final tube of resuspended and clean culture contained no more than 4 mL. We then took cuvettes and measured the OD of each culture in a 1:100 dilution. We then calculated the amount of liquid culture we would need to put in each flask to produce an equal starting OD for both flasks of around .4
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Every hour, we took 1 mL samples from both flasks, spun them down, sterile filtered the supernatant, and stored the resulting filtered supernatant for transformations the next day. We also took OD measurements of samples from the flask. The flasks were incubated for an hour at 37 C before being taken out for measurements.
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The results of the experiment indicate that not only do we have successful lysing of the relevant cells, but we also are able to recover the relevant DNA, not only as is found in the supernatant by qPCR but also by looking at the number of colonies produced by each hourly sample when transformed.
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[[File:colony_count_c2lys.png|350px]]
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[[File:mrfpgene_in_supernat_c2lys.png|350px]]
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[[File:od_lysis_invivo.png|350px]]
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== Thursday, 15 September 2011 ==
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Lilia prepared pSB1C3 backbone from distribution KitPlate in linearised form and also transformed it DH5alpha. If the transformation works, minipreps on pSB1C3 will be done tomorrow.
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4x1L of LB-agar were prepared and should be autoclaved by tomorrow in AI. LB-kanamycin plates are ready and will be brought to BM.
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[[File:EPFL2011_muTetRs_characterisation_table_15.09.11.png|700px]]
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Nadine tested 5 colonies on each Gibson assembly plate, but with no results... So Gibson need to be done again, this time purifying the TetRs before. She also digested the Plac biobrick fragment with EcoRI and PstI.
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== Friday, 16 September 2011 ==
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Lilia made colony PCR on muTetR variants from the 2nd batch of biobricking: TetR_Biobrick_f &_n primers and EPFL taq polymerase were used.
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[[File:EPFL2011_muTetRs_biobricks_2nd_batch_16.09.11.png|600px]]
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[[File:EPFL2011_muTetRs_biobricks_2nd_batch_16.09.11_part2.png|600px]]
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The same PCR on plasmids from the first batch of biobricked mutants gave an unexpected band at higher MW than what was expected. Same for each of five mutants. 
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[[File:EPFL2011_muTetR_biobrick_1st_batch.png|250px]]
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The pSB1C3 biobrick backbone was linearized by PCR with the pSB_prep_2Eb and pSB_prep_3P-1 primers, but the PCR failed - there is no plasmid band in the second well on the gel. It was also transformed into DH5alpha on CM LB-agar plate, but that failed because pSB1C3 in incompatible with DH5alpha cells.
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[[File:EPFL2011_pSB1C3_PCR_linearised_plasmid_backbone.png|250px]]
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Nadine ligated the Plac biobrick into pSB1C3 and transformed. She also purified the TetR genes with Gibson extensions, sending them for sequencing after having done a new Gibson reaction. The Gibson reactions were also transformed, hoping that this time it will work.
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Second attempt to PCR tetR mutants with BioBrick primers yield in PCR product for all the mutants we have:
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[[File:EPF-Lausanne2011_Gel_mutagenesis.tif|600px]]
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We will now digest and ligate it to the pSB1C3 backbone!
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== Saturday, 17 September 2011 ==
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Nadine made colony PCRs on the Gibson assemblies with TetR mutants and on Plac-biobrick ligations. For the Plac-biobrick, all the 5 colonies tested seem to have the Plac insert.
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Doug made all the graphs of Nadine's platereader experiments. We still have some platereaders left to do: J61002 Ptet-RFP wil be done tomorrow and hopefully the TetR mutants will be ready and co-transformed on Tuesday.
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{{:Team:EPF-Lausanne/Templates/Footer|title=Notebook: September 2011}}
{{:Team:EPF-Lausanne/Templates/Footer|title=Notebook: September 2011}}

Latest revision as of 08:43, 21 September 2011

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