Introduction
1. Design of Complex Promoter
In 2014 iGEM competition, our team (SCU-China) designed and submitted a regulatory complex promoter biobrick BBa_K1375024. This part is designed to be a bidirectionally regulated promoter, which can be induced by C4-HSL, and repressed by C12-HSL. To achieve this goal, we used a 53bp long pRhl (BBa_R0071) as template (Fig.1). The RhlR binding site is upstream from the -35 element, which has 3bp overlap with the binding site. We inserted a LasR binding site (ATCTATCTCATTTGCTAG) into the space between -35 element and -10 element of pRhl. Meanwhile, we changed the sequence of original -10 element into standard sequence TATAAT in order to improve the efficiency of initiation of this promoter. The final sequence of this part are shown as below:
5’-GTGAAATCTGGCAGTTACCGˇATCTATCTCATTTGCTAGˇTTATAATGTGTTC-3’
Fig. 1. The sequence of pRhl and RhlR binding site
2.Verification
In order to test the regulatory efficiency of our complex promoter, we constructed the following testing circuit with reporter gene blue chromoprotein amilCP (BBa_K592009)(Fig. 2.) :
Fig. 2. pRhl/Las testing gene circuit
Result
1. Inducement testing with C4-HSL
The circuit was transformed into E.coli strain BL21 (TaKaRaTM). After normal culture for 12h (37oC, 200rpm), we separated bacteria into 4 tubes with C4-HSL concentration of 10μM, 100μM, 1mM, 10mM respectively. After 4h and 16h inducement, the results are shown as follows (Fig. 3):
Fig. 3. Pre-experiment results of C4-HSL inducement after 4h (Top) and 16h (Bottom)
It shows that within certain extents, the efficiency of initiation of our complex promoter is positively correlated with the concentration of C4-HSL and the time of inducement. In order to explore the exact relationship between the concentration of C4-HSL and protein expression, we set a more continuous concentration gradient.
The information of amilCP (BBa_K592009) shows the protein has an absorbance maximum at 588 nm wavelength, we decided to measure the average protein production per unit bacteria by measuring the value of OD588/OD600, in which the wavelength 600nm is used to measure the bacteria concentration. Thus, we set a concentration gradient (0uM, 10uM, 100uM, 200uM, 300uM, 400uM, 500uM, 600uM, 700uM, 800uM, 900uM, 1mM) with 6 parallel test group for each concentration. After incubating for 4h (37 oC), we measured the value of OD588 and OD600 of each group with spectrophotometer (UV-VIS TU1800). We then calculated the average value and the ratio of OD588/OD600. The results are shown as follows (Fig. 4):
Fig. 4. Spectrophotometric results of concentration gradient experiments of C4-HSL inducement.OD588 for reporter protein concentration detection(Top). OD600 for bacteria concentration detection(Middle). OD588/OD600 for average protein production per unit bacteria(Bottom).
According to the data, the value of OD588 and OD588/OD600 show weak correlation with the concentration of C4-HSL. But the lightness-darkness gradient of bacteria colony after centrifugation was obvious (Fig. 5):
Fig. 5. Gradient concentration of C4-HSL inducement results. Every 6 tubes of each column are identical test groups. The concentrations of C4-HSL are 0uM, 10uM, 100uM, 200uM, 300uM, 400uM, 500uM, 600uM, 700uM, 800uM, 900uM, 1mM (from left to right).
According to the figure, the concentration of C4-HSL and protein expression are still positively correlated. We speculated that the reason why spectrophotometry didn’t show expected results is that the wavelength of 600nm and 588nm are so similar that the interference between each other are too severe. So there should be some other methods to get more accurate data.
2.Repression testing with C12-HSL
To test the repression effect more accurately, we used Rhl and Las simultaneously. We set tube 1-8 (Table 1) to observe the amilCP expression.
Table 1. Inducement/repression efficiency test
The results are shown below (Fig. 5)
Fig. 5 The results of Inducement/repression efficiency test.A. After 4 hours, there is no abvious phenomenon.B. After 16 hours , the results were just as we expected. The more Las molecule we added, the stronger repression it showed. C&D. Things went different with the 7th tube after over 20 hours. It showed unexpected blue color, as deep as the 8th tube. This may be due to the crosstalk between Rhl and Las.
According to the Inducement/repression efficiency test results, the repression was effective, except of the results of tube 7 in inducement/repression test after 20 hours, in which the unexpected blue color emerged. We speculated that the reason may be due to the crosstalk between small molecule Rhl and Las. Further tests are still required.
Conclusion
According to the results shown above, PRhl/Las was successfully induced by Rhl and repressed by Las, which means the function of PRhl/Las is as expected. Thus, we successfully improved the characterization of BioBrick PRhl/Las. References
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