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Team:Warsaw/Calendar-Main/17 July 2009
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Assembly of endosomal detection operon
Marcin
Task 1:
- Ligations of biobricks
Comment:
Due to obtain set of biobricks which each of them contain RBS and particular coding sequence I designed the following elemets (the order of biobricks is from the 5'end to 3' end):
- BBa_B0032+BBa_c0040
- BBa_B0032+BBa_C0051
- BBa_B0032+BBa_E0032
- BBa_B0032 (ligation of empty vector as a negative control of the experiment)
Methods:
- Ligation mixture composition: 3 μl digested plasmid with RBS, 3 μl digested CDS, 5 μl ligation buffer (Fermentas, PEG4000 have been added previously), 1 μl ligase T4, 13 μl MQ water
- Negative control mixture composition: 3 μl digested plasmid with RBS, 5 μl ligation buffer (Fermentas, PEG4000 have been added previously), 1 μl ligase T4, 16 μl MQ water
- Duration of ligation was about 12 hours
Task 2:
- Transformation of chemocompetent E. coli strain DH5α
Methods:
- Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes
- Detailed protocol of ligation is described here. The only modification is total volume of ligation mixture prepared 16.07.09
- petri dish were hold in 37°C for 36 hours
Cloning of p53 coding sequence
Marcin
Task:
- Isolation and evaluation of pKS/p53 plasmid
Comment:
Besides high effectivity of transformation all of the bacterial colonies were blue. It is possible that excess of X-Gal may cause some problems with the selection system. Due to this uncentainty I decide to prepare a few bacterial breedings to isolate the plasmids and digest them.
Methods:
- Prepare LB medium with kanamycin
- Add 3.5 ml of the medium to the probes
- Add one bacterial colony to each probe
- Breed the bacteria about 8 hours
Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2
Franek
Jarek
Jarek and Franek run the gelAnia
only described it. Thank you guys:)Tasks:
- Samples of ligated[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid cut with EcoRI and PstI were run on the gel.
Results: Obtain results confirm ligation of the http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor], the undigested plasmid is visible on the gel. It is probably due to incorrect digestion buffer concentration.
Testing different E. coli strains regarding [http://partsregistry.org/Part:BBa_R0010 lacI] and [http://partsregistry.org/Part:BBa_R0080 AraC] repressors
Franek
Tasks:
- Seting new liquid cultures to obtain of pure [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] construct
Methods:
- 2 test tubes with 5ml LB and ampicillin were inoculated with colonies containing [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were left for overnight incubation at 37°C. This time cultures were taken from sparse plate
Results:
- Will be determined next day by cell presence in tubes
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