Difference between revisions of "Part:BBa K4814015"

Revision as of 00:30, 12 October 2023

RecA(K3)-B0034-eGFP

Hoping to validate previous iGEM team’s result and investigate the impact of the genotoxicity of carcinogens, we conducted dosage dependent tests on the top10 E.coli bacteria. RecA(K3), used with eGFP (composite part BBa_K302000) as bioreporter to detect DNA damaging agents such as UV, H2O2 and nalidixic acid, is the promoter derived from BBa_K3020001 by team BIT 2019. This promoter is an optimized version of BBa_K629001 (K6), which is a design of team SYSU 2011 aimed to drive the motor system under a radioactive environment.

We first treated the bacteria and transferred them into 96-well plates to record the fluorescence and optical density (absorbance at 600nm). However, as we discovered that this method might not be accurate enough, we chose to stabilize them on the microscope slides and took photos of the E.coli. After we processed these images, we calculated the mean value of any completely visible bacteria and used the data points from three independent experiments to plot the following box charts.

As can be shown in the following figures, the RecA(K3)-B0034-eGFP design illustrates the dosage dependence.

We then use Fluorescence over OD600 (FL over OD) to compare the EGFP signal in different groups. According to BIT 2019 (https://2019.igem.org/Team:BIT/Bio), SFU is used to compare fluorescence. (Specific fluorescence units SFU=RFU/OD600) After we compared the data of three independent experiments, we can observe that in both UV and H2O2, the SFU is directly proportional to the intensity/concentration of the carcinogen. In addition, the performance of K3 is better than K6, showing that the optimized promoter K3 reduced the background noise considerably.

We also tried different RBS (strong and B0032) to replace the B0034 (BBa_K4814015), and the result indicated that RecA(K3)-eGFP with B0032 RBS has a stronger SFU (FL over OD600) than the original B0034 we chose. Please refer to BBa_K4814002 (B0032) and BBa_K4814013 (Strong RBS) for details.

RBS test of UVB and H2O2 FL over OD600 of RecA(K3)-B0034-eGFP, RecA(K3)-B0032-eGFP, and RecA(K3)-Strong-eGFP.

RBS test of Nalidixic acid and Aspartame FL over OD600 of RecA(K3)-B0034-eGFP, RecA(K3)-B0032-eGFP, and RecA(K3)-Strong-eGFP.

Sequence and Features