Endogenous Induce&Resistance Selection

Here, we want to test the resistance against tetracycline of Alk-Sensor induced by endogenous alkanes.

We put the alkane producing module into cells to test the function of TetA:

Construct:

Left: No.60: TetA downstream of Palkm and AlkR downstream of J23103, are cloned into vector PSB1C3 and there’s a alkane producing vector PSB3K3.

Right: No.61: We construct a blank vector PSB3K3 without NPDC and AAR as blank control, and the other vector PSB1C3 is the same as No.60.

Characterization:

Strains of No.60 are cultured in LB medium for about 12 hours. Add 17μg/mL Kan, 12μg/mL Cm and 12μg/mL Tc respectively. After that, inoculate into 3 ml LB medium for an overnight cultures at 37 ℃ with 220rpm shaking.

Result:

Alkane producing E.coli can grow in tube but blank vector E.coli cannot grow .

Conclusion:

Synthesized alkanes induce the expression of alkR, which enables E.coli to survive under the pressure of Tetracycline.

Thus, we can get a 3×3 matrix, with promoter strength from weak, medium to strong, and plasmid copy number from small, medium to large.

Library Construction

In this part, we will use our constructed Alk-Sensor to help us modify the alkane synthesis pathway. It’s reported that NPDC is a key enzyme in alkane synthesis pathway, but its activity is very low, so we choose NPDC gene (fatty aldehyde decarboxylase derived from Nostoc punctiforme PCC73102) as our mutation target.

We use common ep-PCR method to conduct directed modification to NPDC and construct the mutation library. Alk-Sensor can be used to realize directed evolution. In the alkane producing module we constructed, we introduce restriction enzyme cutting sites, SacⅠand BamHⅡ to both sides of NPDC, to make our manipulation easier.

We use traditional PCR to conduct the process of fast PFU to amplify the segment of NPDC.

Then, we conduct ep-PCR to this segment for 30 cycles, and construct two kinds of strains one with this segment under the regulation of J23114 on PSB3K3 vector and the other under the regulation of J23100 on PSB1C3 vector. We transform these two strains into TranT1 highly-efficient competent cell. We amplify the plasmids and extract whole plasmids. Therefore, we can get a library of strains with different alkane productivity.We picked 20 colony,after sequencing we find that in the 700 base group,54 sites are mutated.And a total of 80 mutations