Yesterday, our reporter circuit with GFP:LVA (
Pqrr4+B0034+K082003) was constructed and transformed using colony 1 of
Pqrr4+B0034 and already cut and freshly cut K082003. The K082003 part was already cut last time I attempted to construct this circuit, and this was used as well as a freshly cut K082003 to see if there are any differences between the two. On the plate of
Pqrr4+B0034+K082003(old), there were noticeably less colonies than on the plate of
Pqrr4+B0034+K082003(new). Perhaps most of the old K082003 linear DNA has degraded and was not cloned into
Pqrr4+B0034, and most of the phosphotase treated vector remained as a linear piece of DNA, meaning the cells could not take up the plasmid that contains the antibiotic of interest. The following is the picture of the gel:
The band in (C1, old), (C3, new), (C4, new), and (C7, new) lanes seem to be at the expected size, at around 1170bp, including the additional bps that are due to the annealing of the Biobrick CP reverse primer. I will procede with these colonies tomorrow with Restriction digest to further verify the presence.
Today, I played around with the plate reader. Blank LB broth with Kanamycin and Chloramphenicol antibiotics media was used to blank the machine. The following was the result:
A1~A6 consists of the results from TOP10 cells with the reporter and mutant circuits. A7~A8 constists of the results from KT1144 cells with mutant circuits in them. According to http://www.mnstate.edu/provost/GFPPlateReaderAssayProtocol.pdf, the settings for the plate reader should be:
Excitation: 485/20 nm
Emission: 528/20
Just by looking at the results, it seems that the cells indeed do produce GFP. However, since I am yet to know what values I should be expecting to conclude that they are producing GFP, I cannot be sure; thus I will explore some papers in order to find some data to compare with.
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