Difference between revisions of "Team:USTC/Results"

We got the genes: Promotor j23100, RBS B0034, luxR C0062, luxI C0061, lux pR R0062 from the iGEM Distribution Kit. Then we assembled the standard Bio-brick using the four restriction enzymes: EcoRI, XbaI, SpeI, PstI. We performed double digestion on JTPI plasmid using EcoRI and PstI, which were only on the plasmid backbone, and the result shown in Figure 1. From the result of electrophoresis, we confirmed the of JTPI.

Considering that the positive feedback may make our system unstable, we constructed another plasmid without LuxI gene, and the result of double digestion on JTP shown in Figure 1.

Figure 1. Electrophoresis result of double digestion of JTPI and JTP plasmid.

We confirmed our plasmids by genetic sequencing in Sangon Biotech afterwards.

Since the data from modeling simulation indicated that the copy number of the plasmid also made an impact on the feasibility of our system, we constructed the same system on the pET28b. We amplified the Regulation System from the JTPI plasmid and the pET28b plasmid backbone, then we assembled two gene fragments using the Seamless Cloning Master Mix. We transformed the plasmid into E.coli(BL21).

In order to verify the function of our system, we constructed the plasmid combined the regulation system and GFP. From the result of electrophoresis shown in Figure 2, we confirmed that JTPIG and JTPG were successfully constructed.

Figure 2. Electrophoresis result of double digestion of JTPIG and JTPG plasmid.

To figure out the impact of gene LuxI and different plasmids on the function of our system in E.coli. We measured relative fluorescent intensity (RFI) and OD600 at various time respectively. Then we plotted the scatter graph and drew the RFI curve and growth curve in B-spline (shown in Figure 4 and Figure 5).

Figure 4. RFI curve of E.coli with JTPI/JTP

Figure 5. growth curve of E.coli with JTPI/JTP

According to the graph of RFI curve and growth curve of E.coli with JTPI/JTP, we can reach these conclusions:

(a) JTPI on pSB1C3 is a plasmid constructed by inserting JTPI into pSB1C3 with GFP downstream. The RFI curve of JTPI on pSB1C3 indicated that the leaky expression of LuxI in JTPI on pSB1C3 was too severe, resulting in high expression of GFP. At the same time, the growth curve showed that the bacterial density was lower compared with others, indicating too much growth pressure of E. coli because of the leaky expression of LuxI. For these reasons, inserting JTPI into pSB1C3 was not a good solution for controlling expression leakage.

(b) JTP on pSB1C3 is a plasmid constructed by inserting JTP into pSB1C3 with GFP downstream. The RFI curve of JTP on pSB1C3 indicated that the low expression of green fluorescent protein was due to the absence of LuxI gene. Meanwhile, the growth curve indicated that the growth pressure of E. coli was not so severe, so bacterial density was higher than JTPI on pSB1C3.

(c) JTPI on pET28 is a plasmid in which we inserted JTPI into pET28 with GFP downstream. As far as we know, the copy number of pET28 is less than the copy number of pSB1C3. The comparison of the RFI curve of JTPI on pET28 with JTP on pSB1C3 indicated that it can meet the needs of our regulation system. At the same time, the growth curve of JTPI on pET28 was not much different compared with the growth curve of JTP on pSB1C3, so the growth pressure of E. coli was not so severe. For these reasons, we can decrease expression leakage via inserting JTPI into pET28.

In summary, we decided to insert JTPI into pET28 and we finally successfully constructed this plasmid.