Difference between revisions of "Part:BBa K1104244:Experience"

iGEM2019[http://2019.igem.org/Team:BIT# BIT]

iGEM2019 [http://2019.igem.org/Team:BIT# BIT] Expression of BBa-K1104244 in trans5α strain and induction of hydrogen peroxide

We introduced BBa--K1104244 into tran5α competent cells, applied them to LB plates containing 300 μg/mL chloramphenicol, inverted overnight at 37 ° C, and then picked monoclonal colonies up into chloramphenicol-resistant LB solid medium overnight. Then take 400 μL of bacterial solution inoculated in LB medium at 37 ° C 130 r / min shaker shaking culture for 2-3 hours, when their OD600=0.2-0.4(Logarithmic period), adding different concentrations of hydrogen peroxide, induced at 37 ° C 130r / min for 2 - 3 hours to generate GFP. We conducted two sets of experiments, and set different concentration gradients to observe the induced expression of GFP.

After being induced at 37 ° C 130r / min for 2 .5 hours, add 200 μL bacterial solution into a black 96-well plate (Corning, USA) for fluorescence determination. We set the fluorescence excitation wavelength to 488 nm, and the emission wavelength to 516nm, and the OD600 was measured at the same time.The two sets of data were obtained by scanning at room temperature in a multi-function microplate reader. The data obtained is as follows:

First set:

The promoter strengths are standardized by setting AhpCp2d1 in the absence of H202 as 1

specific fluorescence units：SFU=RFU/OD600

Second set:

The promoter strengths are standardized by setting AhpCp2d1 in the absence of H202 as 1

specific fluorescence units：SFU=RFU/OD600

It can be seen from the above experiment that within a certain range, as the concentration of hydrogen peroxide increases, the amount of fluorescent expression induced by the promoter-induced expression increases. This is basically consistent with the results obtained by the iGEM13_NYMU-Taipei (2013-09-16) team.