Meeting
9:30 - 11:00
Jakob, Julia, Manuel, Rüdiger, Sandra, Sophie, Theo
red light parts
- after the test digest and the sequencing the next step would be to clone the parts behind the promotor-RBS
green light parts
- after the test digest of Ccas and CcaR and the sequencing the next step would be to clone the parts behind the promotor-RBS
blue light parts
- after the test digest and the sequencing the next step would be to clone the parts behind the promotor-RBS
precipitator
- the gene sequences will be sent to ATG biosynthetics today
plastic binding domain (PBD)
- the GFP was amplified via PCR and primers that contain the PBD, unfortunately the primers contained restriction sites for XbaI and SpeI
- next step would be to take an plasmid with promotor-RBS and open it with SpeI, then combine this with the GFP-PBD and select enough colonies to catch a plasmid with the insert in the right direction (50:50 chance)
lysis part
- the part we got after cloning the repressor behind a promotor-RBS is missing the scar after the RBS according to the sequencing results (6 bases)
- next step would be to correct this error, possible solutions: large primers to amplifie the whole construct, short primers to amplify the repressor only, mutagenesis of the missing 6 nucleotides, solve it via gibson cloning
other stuff
- notbook entrys should be updated from now on to the wiki
- twitter should be updated (movie, etc..)
- a weekly description of the accomplishments should be prepared
- project description and safety sheet should be discussed on monday
- on tuesday we visit the NGS presentation from Eurofins
- we need a printer in the lab: Sophie wants to bring one from home, if this doesn't work Rüdiger will get one till tuesday
- Rüdiger prepared some SOPs: gelextraction, miniprep, documentation of gels, you can find them on the snapserver in the folder iGEM/SOPs
- Sandra prepared the 3A Assembly, Transformation SOP
- still missing are: PCR, test digest, transformation, agarose gel electrophoresis
Test-digest and Gel
Jakob
Digestion and then a gel was loaded.
- S15a pSB1AK3
- S15b pSB1AK3
- S12a pSB1A2
- S12b pSB1A2
- S11a pSB1A2
- S11b pSB1A2
- S17b pSB1C3 (Ccar)
- M18b pSB1C3 (Ccar)
- S24 pSB1C3 (pcyA and terminator)
- S25 pSB1C3 (pcyA and terminator)
- S26 pSB1C3 (pcyA and terminator)
- S27 pSB1C3 (pcyA and terminator)
- S28 pSB1T3 (cph8)
- S29 pSB1T3 (cph8)
Precipitator
Digestion of PR vector
Investigators: Sophie, Rüdiger
PR-vector: pSB1C3 (2070 bp)
Ligation of GFP-Pbd in PR-vector
Investigators: Rüdiger
- Digestion of GFP-pbd with xbaI and SpeI
- Digestion of PR-vector with SpeI
Then ligated and stored with the names M2 and M3 in the freezer.
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