Part 0 Construction of High-glutamate-yield B. amyloliquefaciens LL3
In order to get a Bacillus amyloliquefaciens LL3 strain for a higher glutamate yield, we edited the genome by constructing promoters in tandem to overexpress glutamate related genes, icd and gltAB, which are responsible for 2-ketoglutarate and glutamate, separately. Due to the difficulties of genome manipulation, we only succeeded in inserting Pc2up ahead of icd and get a high yield strain.
Part 1 Verification of PgltAB Responding with Glutamate and GltC
In Bacillus amyloliquefaciens LL3, there is a glt operon which is responsible for the intracellular glutamate synthesis. Since we need to use the regulatory role of glutamate and GltC to PgltAB, we need to verify the effect of promoter PgltAB under different glutamate concentrations. So we constructed pHT01-PgltAB-GFP and pHT01-P43-GFP 2 plasmids and transformed them into Bacillus amyloliquefaciens LL3 icd strain. We fermented under different glutamate concentrations and tested the fluorescence intensity(FI) of GFP every 6 hours in M9 medium. For the strain transformed with pHT01-PgltAB-GFP, as the glutamate concentration goes up, the FI of GFP first went up and came down. On the other hand, for the strain transformed with pHT01-P43-GFP, FI is uncorrelated to the glutamate concentration.
Part 2 Verification of the Repression Effect of LacI to Pgrac
To make our circuit work, we need to test the inhibition effect to Pgrac which can respond to LacI protein at different lacI expression levels. So we expected to construct vectors pHT01-LacI-Pdifferent-Pgrac-TetA.
Part 3 Constrction of the Whole PopQC System and Functional Test
Our project is aiming to construct a PopQC system in a single plasmid. So we construct the vector pHT01-LacI-PgltAB-Pgrac-TetA and ferment to test the function of our system. To furthermore simplify the procedures of functional test, we constructed another vector pHT01-mCherry-LacI-PgltAB-Pgrac-TetA-GFP(int) to test the fluorescence intensity instead of mRNA. The results are roughly in accordance with our expectation.
We constructed 12 standard parts, 1. PgltAB-LacI-Pgrac-TetA (BBa_K2705006); 2. LacI(BBa_K2705001); 3. PgltAB(BBa_K2705000); 4. Pgrac(BBa_K2705002); 5. TetA(BBa_K2705003); 6. GFP(BBa_K2705004) 7. PgltAB-GFP(BBa_K2705005); 8. PliaG&43(BBa_K2705008); 9. PliaG&43-1(BBa_K2705009); 10. PliaG&43-2(BBa_K2705010); 11. PliaG&43-3(BBa_K2705011); 12. TetA(optimized)(BBa_K2705003). The promoter PgltAB, Pgrac, lacI, tetA, and gfp genes are cloned by PCR. Then both these segments and pSB1C3 vector were treated with restriction enzymes. After that, these parts were separately ligated to linear pSB1C3 by ligase or recombinase, and the products were separately transformed into E.coli JM110. Verification PCR was performed to select the positive clones. The positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.
We improved 2 parts, BBa_K823000 and BBa_J31006. For BBa_K823000 the weak, constitutive promoter from Bacillus subtilis, we chose to mimic the structure of P43, the relatively strong constitutive promoter which can be recognized by both sigma factor 55 (the major sigma factor A) and sigma factor 37 (the lag phase sigma factor B). After designing the new sequences of promoters, we send them to the company GenScript Biotech Corp. to have them synthesized. The PliaG&43-0, PliaG&43-1, PliaG&43-2, PliaG&43-3 are cloned by PCR. Then both these segments and pHT01-GFP vector were treated with restriction enzymes. After that, these parts were separately ligated to linear pHT01-GFP by recombinase, and the products were separately transformed into E.coli JM110. Verification PCR was performed to select the positive clones. The positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced. Object plasmids are transformed into Bacillus subtilis 168 by electroporation transformation and verification PCR was performed. After that, we use the Microplate Reader to analyze the FI of the cultures after 2 hours. For BBa_J31006, we send the sequence to the company GenScript Biotech Corp. and get a codon optimized one for Bacillus subtilis. And then we constructed pHT01-P43-TetA plasmid and transformed into Bacillus subtilis LL3 to test its function by measuring the MIC.