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_1408_t7.jpg|200px]]
[[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.
Finally, the Plac promoter being correctly inserted into J61002 Plac-RFP plasmid, Nadine also ran a PCR to add biobrick extensions to the promoter.
[[File:EPFL_Igem_1409_PlacBB.jpg|200px]]
[[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]]
== Thursday, 15 September 2011 ==
== Thursday, 15 September 2011 ==
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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.
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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