Difference between revisions of "Team:LZU-China/chz collaborations"

We started our contact with team TJU from the very beginning of the summer. Since both teams are working on the MFC system, we have shared a lot of experience on it, including the MFC device setup, medium condition control and part characterization. Additionally, we had lots of online and offline discussion with them.

In the middle of the project, we came to Key Laboratory of Systems Bioengineering (Tianjin University) to have a project seminar with them. During the discussion, we knew that they have constructed a lactate producing part named ldhE. However, their characterization of this part was faced with some problem. To be specific, engineered E.coli strain bearing the ldhE part showed little difference with the wild type. (shown as follows)

Fig.1 The production of lactate in MG1665-WT. The cells were cultured in 37C for 15h and the lactate concentration was measured by HPLC.

For the lactate producing system, we have helped team TJU to optimize and characterize the ldhE part. From their introduction, we know that LDH is an enzyme catalyzes the conversion of pyruvate to lactate with NADH serving as the coenzyme. Initially, they intended to introduce high-yield u L-(+)-lactate dehydrogenase gene (ldhA) from Lactobacillus, which could produce a relatively larger amount of L-lactate for Shewanella. Consequently, we decided to focus on the assistant method to help them resolve the problem.

After some consultant, we noticed that in the wild type E. coli, LDH reaction is not as competitive as the reaction through PFL, which might result in the ineffectiveness of ldhE.[1] Therefore, knockout of the PFL related genes will contribute to redistribute the metabolic flux. In addition, we searched in the part registry of IGEM and found the part BBa_K341458 and part BBa_K341002, which revealed a more effective and easy-to-use method for gene knockout.

In the system we searched, the cell is first transformed with a helper plasmid harboring genes encoding the λ-Red enzymes, I-SceI endonuclease, and RecA. λ-Red enzymes expressed from the helper plasmids are used to recombine a small ‘landing pad’, a tetracycline resistance gene (tetA) flanked by I-SceI recognition sites and landing pad regions, into the desired location in the chromosome. After tetracycline selection for successful landing pad integrants, the cell is transformed with a donor plasmid carrying the desired insertion fragment; this fragment is excised by I-SceI and incorporated into the landing pad via recombination at the landing pad regions.[2] We think this more effective and easy method would help them overcome the problem. So, we recommended TJU to adopt the two-step λ-Red method to knock out the pflB gene.

After their reconstruction of the engineered strain, a more visible contrast was shown as below.

Fig.2 The production of lactate of the MG1655ΔpflB. The cells are cultured in 37C. (Data from TJU)

In order to prove the cell can work normally, we tested the knock-out cell’s field of lactate in our lab. The result is shown in the figure below.

Fig.3 The production of lactate of the MG1655ΔpflB. The cells are cultured in 37C for 26h. (data from LZU-China)

References

[1]Zhu J, Shimizu K, Zhu J, et al. Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition[J]. Metabolic Engineering, 2005, 7(2):104–115.

[2]Thomas E, Kuhlman, Edward C, Cox. Site-specific chromosomal integration of large synthetic constructs.[J]. Nucleic Acids Research, 2010, 38(38).