BUCT pursues preciseness and efficiency in the process of exploring problems. We are not afraid of failure. After experiencing failure, we will continue to review, discuss, draw lessons and strive for higher achievements!

BUCT's exploration process for problems can be roughly summarized into three cycles, cycle 1, 2 and 3. In each cycle, we will continue to move forward in the way of try face the problem solve the problem further try.

Degradation of creatinine

Cycle 1 Cycle 2 Cycle 3
We used Pseudomonas putida NBRC 14164 preserved in the laboratory for resuscitation culture, and extracted the whole genome of the bacterium. It was found that the fragment could not be amplified by conventional methods. We found that the reason might be the inappropriate annealing temperature. Therefore, we tried to use the method of cyclic heating up. After continuous attempts, we amplified crnA by touch down PCR, and ordered the gene fragment of crej, After homologous recombination on pCS27-lac plasmid, pCS27-lac-crnA and pCS27-lac-crej-were obtained, which were correct after sequencing. The expression frame in pCS27-lac-crnA was amplified separately (with SacI and BcuI digestion sites) and connected with the digested pCS27-lac-creJ under the action of T4 ligase, but the transformation efficiency was not high. Therefore, we amplified the two expression frames separately, connected them by overlap, homologous recombination with the pCS27 plasmid bone frame, and transformed them after sequencing without error. The fermentation experiment was carried out, and good results were obtained. After the effect of creatinine degradation was successfully verified, considering that crnA and creJ have different reaction rates, we combined them into an expression box and adjusted their arrangement order. In order to reduce the burden of bacteria, we integrated the glutamate racemase coding gene with the same expression intensity into the same plasmid to construct a more complex coexpression plasmid pCS27-lac-crej-crnA race. We learned from the previous lessons, connected each expression frame by overlap, and then homologous recombination with the plasmid backbone. Finally, we sequenced the obtained plasmid, and the results were correct.

Degradation of urea

Cycle 1 Cycle 2 Cycle 3
We first purchased Provincia rettgeri strain jnb815 from CICC and obtained its whole genome. The urease gene cluster was obtained through the primer sequences provided in the corresponding literature. Through data investigation, we found that ureEFGD, an auxiliary gene of urease, is not soluble. Therefore, we tried to solve or improve this problem. First, we linked it to pET duet plasmid, transformed it into E. coli BL21 to induce protein production, and then broken it. SDS-PAGE verified that groES/EL has a certain effect. We constructed pETlac-ureABC-lac-ureEFGD and carried out the addition experiment of urea degradation, but the results were not ideal. In order to enhance the effect of ureEFGD, we replaced the lac promoter, so we constructed pze12-plpp1.2-ureEFGD, amplified its expression frame, and constructed pETlac-ureABC-plpp1.2-ureEFGD. The fermentation experiment was carried out again, and the results were slightly improved. Similarly, in order to reduce the burden of bacteria, we integrated polyglutamic acid synthase with the above modules, and finally constructed a more complex coexpression system pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCAE

Synthesis of polyglutamic acid & a complete pathway

Cycle 1 Cycle 2 Cycle 3

First of all, we need to enhance the precursor supply of glutamate, the substrate of this process. We obtained the coding gene of glutamate dehydrogenase in the genome of E. coli K12, and used crispr-cas9 technology at the ygay site to integrate the genome of EcN. However, when the pcas9 plasmid was eliminated, it could not be effectively eliminated at 42 ℃ for 12h. Therefore, we extended the time to 24h and achieved success.

After that, we ordered the gene sequence of glutamate racemase (race) and obtained the gene cluster pgsBCAE of polyglutamic acid synthase in the genome of Bacillus subtilis strain a-5, and constructed pETlac-pgsBCAE, pCS27 lac race. After sequencing, it was correct.

The above two plasmids were co transformed into the chassis to carry out the fermentation experiment of glutamate addition (because the work of gene integration was not completed at this time), and good results were obtained. In the verification of the complete pathway, we constructed two more complex coexpression plasmids pCS27-lac-crej-crnA-lac-race, pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCAE, and completed the verification of fermentation with gdhA integrated Escherichia coli Nissle 1917 as the chassis.

Suicide system

Cycle 1 Cycle 2 Cycle 3

Our initial idea was to build a temperature sensing suicide system, using ci857 as a key element, but after our discussion, this element lacked enough innovation to help future teams, so we abandoned it.

Later, we decided to use the difference of ammonia nitrogen concentration to build an innovative biosafety system. We localized glnAp2. After our characterization, the promoter can show different expression intensity due to the change of external ammonia nitrogen concentration, but the intensity change it shows cannot meet our actual needs. Therefore, mutation of this sequence has become our main task.

Fortunately，we screened about 1600 samples in total, and finally found these four samples in all mutated samples, which is a considerable workload for BUCT, which is completely composed of undergraduate students. After obtaining the correct mutated samples, we sequenced them. We successfully used this element to construct a suicide system with bsrg/sr4 as toxin antitoxin and ttgr PTTG as repression system (we obtained them in the genomes of Bacillus subtilis and Pseudomonas putida), and verified the feasibility of the suicide system under the induction of different concentrations of ammonia nitrogen.