Team:Paris Bettencourt/Notebook/Indigo
Week 27th June - 3rd July
Week 4th - 10th July
We received the E. coli BL21 and BL21(DE3) - same background, but BL21 does not express the T7 polimerase and BL21(DE3) does.
Highlights
Transformation efficiency: 1–5 x 107 cfu/μg pUC19 DNA
Routine non-T7 expression
Deficient in proteases Lon and OmpT
Resistant to phage T1 (fhuA2)
B Strain
Free of animal products
Genotype
fhuA2 [lon] ompT gal [dcm] ΔhsdS
Week 11th - 17th July
This week we started by plating the BL21 and the BL21(DE3) strains and making competent cells out of them.
We transformed E. coli DH5alfa BBaa_K863000 (bpuI plasmid).
We minipreped the pbul plasmid using the ThermoScientific GENEJET Plasmid MIniprep kit #K0503
Protocol was missing.
https://tools.thermofisher.com/content/sfs/manuals/MAN0013117_GeneJET_Plasmid_Miniprep_UG.pdf
We electroporated 2uL of plasmid and 20uL of the electrocompetent cells. We plated on LB crm25, 200uL and 500uL (Jake told us that 99% of the cells would die due to overexpression of our protein)
Confluent growth observed on yesterday's plates (problem with antibiotic? - I do not believe so because the plate with the + control - interstudy + control with the GFP - shows that only cells carrying the plasmid were able to survive on the plate - See Figure)
Restricking the laccase plasmid ones just in case in new plates.
Week 18th -24th July
Week 25th -31th July
+33 1 44 41 25 22/25
igem2016parisbettencourt@gmail.com 