Model

Here, we designed a CBE-Mag-RNA polymerase system for do semi-random mutagenesis on GFP and other metabolic related enzymes in cells. CBE-RNA polymerase as a positive control. The negative control is with treatment of no adding IPTG and no treating with blue light. As shown in projects-results Figure 1-7 part and engineering success part, we confirmed the success of our semi-random mutagenesis system by CBE-Mag-RNA polymerase using Sanger sequencing and Next generation sequences.

But there is one important question remained. How long shall we need to irradiate the bacteria with the blue light. In order to qualify the mutation rate and the time of blue light irradiation. Mathematical modeling is needed for testing the relationship. The overall question is when shall we need to stop the blue light to make the mutagenesis system happened in the reasonable way.

1. The first relationship is blue-light irradiation time and mutation rate?

To test this question, we have performed a 0, 2 ,4, 6, 12, 24 hours irradiation time treatment to E .coli with CBE-Mag-RNAP or CBE- RNAP or No IPTG (negative control). Based on the liner regression, CBE-Mag-RNP is Y = 5.015*X - 4.622 while CBE-RNAP is Y = 5.435*X - 0.2608 indicating the mutation happen 5 times in every 1000 base in every hour. More importantly, when the blue light is on, the blue light dependent CBE-Mag-RNAP system and the blue light independent CBE-RNAP system are nearly working in the same efficiency, indicating that p-Mag and n-Mag has strong combination under the blue light

2. The second relationship is sufficient bake when without blue-light irradiation and mutation rate?

To test this question, we give the CBE-Mag-RNAP or CBE-RNAP or No IPTG (negative control) three group with 24 hours blue light irritation, chasing by dark treatment for extra 1, 3, 5, 8 hours. We performed DNA sequencing and count the C to T in every 1000 base. Based on the liner regression, CBE-Mag-RNP is Y = 3.660*X + 123.8 while CBE-RNAP is Y = 17.49*X + 118.4 indicating CBE-Mag-RNP mutagenesis became slow in dark condition while CBE-RNAP, a positive control, keep editing and become faster to 17 base/kb/h speed as the CBE-RNAP accumulation in the cells.