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 the wild type E. coli's 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 production 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).

SYHS team is a young but rich prospects team. SYHS, a high school with the long history, is one of the best high school in three northeastern province of China. This is the first year of them to join the iGEM competition. Although they attend iGEM as a high school team, it is not only a training but a challenge as well. However, working as a team lack of experience and knowledge, they have faced some problems. Therefore, in the spirit of helping others, we gave them a lot of advice in presentation, the wiki design and the parts construction.

For example, their use of genetic engineering techniques are not skilled, so we helped them in the field we familiar such as selecting the clone and assembling the plasmid.

What’s more, we helped them about the adaption between mediawiki platforms with html. We pointed an important mistake for them, which made them avoid adding a wrong part and told us many things about the part shipping. They completed the part submission form with our assistance. We helped to choose the suitable Part ID and told them how to fill in the Registry.

What SZU-iGEM have done this year was to create an anti-bladder-cancer-cell system which is regulated by an AND GATE. What we had promoted was to put the effector gene Renilla Luciferase to replace Firefly Luciferase in the third plasmid which has the strong constitutive promoter SV40. The remould was supposed to increase the security of the AND GATE and the expression degree of the system, and together the influence of the orthogonal system to the project system.

1. Security testing

After transforming the three plasmids system into T24 cells for many times, the result of expression examination showed the system had a nice security degree. Compared to the two plasmid system in Hela cervical cancer cells, the expression of the three plasmid system in the control group without Ack has the same level. While the expression of the three plasmid system is expected to improve the expression of the project system when the Ask exists, the examination showed that our remould has no effect on the security of the system.

2. Effect testing
2.1. Examining by Rlu

Comparing with the expression of the group without Ack, adding Ack has reached more than 7 times. It shows that the effect of orthogonal system on the whole system has been improved. In addition, the average expression of adding Ack experimental group has reached 790.5, and in others successful transfection experiments, the highest number of test results have reached 1000. All the data indicated that expression amount of the system has enhanced a lot after the remould.

Fig.1 The result of system verification after remoulding

Rlu:Relative light unit

No ACK in T24:Representing plasmids were transfected into T24,without adding ACK

Add ACK in T24:Representing plasmids were transfected into T24,with adding ACK

2.2. Examining by GFP

After replacing Rlu with GFP, we transformed two plasmids system and three plasmids system into T24 bladder cancer cells respectively. The positive control group is T24 with a plasmid which carried normal GFP behind CMV promoter. The result we got using Leica fluorescence microscope photographs, which showed that the three plasmids system do have a higher expression degree than the two plasmids system as follows:

Fig.2 The result in T24 of positive controll group

Fig.3 Experimental group without ACK(two plasmids system)

Fig.4 Experimental group adding ACK(two plasmids system)

Fig.5 Experimental group without ACK(three plasmids system)

Fig.6 Experimental group adding ACK(three plasmids system)