Team:XHD-ShanDong-China/Contribution
Contribution
In order to provide useful support and contribution to future iGEM, we have carried out the following three aspects of work:
- ● Characterization of a related previous part BBa_K338001 supplemented with new data.
- ● Provide new data from relevant literature for the previous part BBa_J45006 .
- ● Created a 3D printed model of E. coli chromosome, a chassis creature commonly used in iGEM, and filmed the process of making the model.
Based on BBa_K338001 part, a genetic circuit HSdegPLCP was constructed to characterize the function of HS promoter.
We construct the following gene circuit based on the principles of synthetic biology, as shown in Figure 1. The HS promoter was amplified from E. coli MG1655, the vector pRdegPLCP was linearized by PCR, and then replace the rDegP of pRdegPLCP by HS promoter to obtain pHSdegPLCP by homologous recombination, and the recombinant plasmid was verified by PCR to ensure the success of the recombinant.
Figure 1. Schematic diagram of expression circuit.
The plasmid pHSdegPLCP containing K338001 part was transformed into DH5α, and the DH5α bacteria containing the recombinant plasmid pHSdegPLCP were cultured overnight in LB medium. Subsequently, the new LB medium was inoculated at a ratio of 1:100 and cultured at 37°C at 200 rpm for 2 hours to log phase. Subsequently, 6 tubes of 5mL LB medium were inoculated at a ratio of 1:100, 3 tubes were cultured at 37°C, and 3 tubes were cultured at 45°C. Take 200uL to 96-well plate every 0.5h, and measure the concentration of amilCP with a microplate reader. The concentration of amilCP (OD588) and OD600 value are measured simultaneously.
We used the E. coli MG1655 as template and then performed PCR to obtain the heat shock(HS) promotor fragment. We use plasmid pRdegPLCP as a template to obtain a linearized plasmid vector by PCR amplification. As shown in Figure 2, the size of the PCR product was as expected. Then use Gibson assembly to obtain the recombinant plasmid pHSdegPLCP. The PCR verification showed that the recombination was successful, as shown in Figure 3.
Figure 2. Gel electrophoresis of HSP and linearized pRdegPLCP PCR product.
Figure 3. Gel electrophoresis of PCR verification.
The map of plasmid pHSdegPLCP obtained by the recombination is shown in Figure 4. It contains a chloramphenicol resistance gene, and HSP can activate the expression of degP and amilCP.
Figure 4. plasmid map of pHSdegPLCP.
The concentration of chromoprotein is obtained by dividing the OD588 of the bacterial solution by OD600. As shown in Figure 5, the chromoprotein amilCP shows different expression levels at different temperatures. It can be seen from the figure that starting from the second hour, the chromoprotein produced per cell at 45°C is significantly higher than 37°C. It shows that the heat shock promoter is activated at 45°C, which makes the DegP fused with chromoprotein to express at a high level.
Figure 5. amilCP production per cell mass unit.
Centrifuge the bacterial solution that has grown to stage phase is shown in Figure 6. The one on the left is 37°C and a slight blue color can be seen, and the one on the right is 45°C and a clear blue-violet can be seen. It shows that the expression of blue protein is more at 45°C, that is, HSP activates transcription more activity at high temperature.
Figure 6. Comparison of the color of bacterial liquid at different temperatures.
The DH5α_pHSdegPLCP strain cultured at 37°C to the logarithmic phase was inoculated into 6 tubes of LB medium, 3 tubes were cultured at 37°C, and 3 tubes were cultured at 45°C. The OD600 was sampled every 1h, and the growth curve was obtained after 11h as shown in Figure 7. In a high temperature environment, the growth of E. coli itself is relatively slow, which is significantly lower than the 37°C curve. In this picture, we can see that the growth of E. coli tends to increase after 6 hours. It is because we have connected the degP gene downstream of the heat shock promoter HSP. High temperature promotes HSP to activate a large amount of degP expression. A large amount of DegP protein will increase the survival rate of E. coli. It also proves that the startup effect of the heat shock promoter HSP at high temperature is significantly improved.
Figure 7. Growth curve at different temperatures.
From the results, we can clearly see that in a high temperature environment, HSP can quickly activate the expression of downstream genes.
The acetyltransferase ATF1 one of three known S. cerevisiae alcohol acetyl transferases responsible for the synthesis of volatile esters. In this paper, ATF1 is proved to also acetylate alcohols to make various acetates, which are the main component of moth pheromones. Therefore, we could use this enzyme ATF1 to produce moth pheromone compounds biologically, which could be used in pest control.
In this paper, they decided to determine whether this enzyme was responsible for the background formation of acetates when supplemented with long-chain fatty alcohols. First, they added 19 different fatty alcohol substrates to the atf1∆ knockout strain. The atf1∆ knockout strain produced barely detect- able quantities of any long-chain acetates, suggesting that ATF1 is the only yeast enzyme with significant activity towards fatty alcohols of C10 and longer (Fig. 2). To compare the capacity of ATF1 to that of EaDAcT to produce acetates, we expressed either enzyme in an atf1∆ knockout strain and incubated the cultures with the fatty alcohol sub- strates. Quantification of the acetates formation provided additional evidence that ATF1 can convert a wide range of fatty alcohols into their corresponding acetates (Fig. 2). Further, the levels of acetates formed in ATF1-expressing yeast were 10–40 times higher than those in yeast express- ing EaDAcT, except for 16:OH, 18:OH, and Z9-18:OH which were also poor substrates for ATF1 (Fig. 2).
Based on the existing three-dimensional structure data of E. coli in the literature, we constructed a three-dimensional chromosome model of E. coli and converted it into a 3D printed model.
Figure 8. 3D printed model file.
Figure 9. Printed 3D model of E. coli chromosome.
