The constitutive promoters are widely used in iGEM. Anderson's promoters are the most widely used constitutive promoter family. Those promoters are well characterized by RFP fluorescence. However, a key problem of generalizing the results of characterizations is that the dynamics of gene expression are influenced by the protein coded for. The RFP expression result might not compatible with our gene expression. In 2016 iGEM competition, team William_and_Mary tried to use insulator RiboJ to make relative expression levels similar between different proteins. They have verified this phenomenon as the paper published before. We adopted the same RiboJ insulator design to measure the 10 promoters from Anderson’s Promoters family by expressing eGFP to avoid the influence brought by different coding sequence for our pathway construction.
Why we start to do the measurement is that we need to calculate based on the urate concentration in the gut to evaluate if our pathway is efficient enough to reduce the urate in the gut, or there's unnecessary extra protein to express theoretically. At the time, absolute protein concentration expressed by different promoters should be known. We have to find out the relationship between eGFP quantitive protein concentration and the promoters. And that's the first time in iGEM to evaluate the absolute protein expression of a promoter with the help of insulator to predict the metabolic ability of a pathway. Our measurement provided a prospective for the teams to evaluate the efficiency of the pathway in the future.
To achieve this, we deciede to draw a standard curve of eGFP fluoresence-eGFP absolute protein quantity. We expressed the eGFP-6x his-tag in pET28a driven by T7 promoter. We purified the protein and draw the standard curve successfully in our group A experiment. (See protocols for purified and measurement experiment details).
Group A: Fluorescence curve of eGFP protein purified. Samples were added twice in each experiment to measure in this group. The experiment has been repeated for 5 times.
The result of group A experiment shows good repeatability, so we coordinated all the data of Group A and drew the fluorescence curve. Data shown yellow are abandoned. Statistical results show that the similarity index of fluorescence curve each experiment is 0.84403025081. You can see the details in the modeling.
However, it’s known that the fluorescence of eGFP may quenching after exaction, e.g. it may degrade when stored. And the measurement error may also influence the curve, which may introduce error to the quantitative experiment. To make our experiment more reliable and highly repeatable, we did the Group B experiment. We stored our samples and measured the same sample every 4 days to check the quenching.
Group B: fluorescence curve of eGFP purified at the same time, at an interval of four days.
In a short time, the fluorescence curve showed no significant change if the protein are stored properly. Statistical results show that the similarity index of fluorescence curve at the same time, at an interval of four days is 0.88848598263493528. You can see the details in the modeling. To summarize, the experiment data shows good repeatability and time change has few influence on the curve.
Then, 10 different promoters with the same gene structures (Anderson’s prmoter + RiboJ + B0034 + eGFP + double terminators) plasmid were transformed in E. coli BL21. The bacteria was cultivated in 37 degrees, 220 rpm shaker overnight (14-16h). The fluoresence and OD600 were measured at the same time to exclude the error brought by the bacteria number.
The result shows that the protein concentration expressed by J23100 is about 10 times more than urate concentration in the gut, which is about 2000uM in bacteria cytoplasm. Detailed calculation process can be seen in modeling.
However, the chassis expression ability should be considered. In our experiment, E. coli BL21 is the chassis for measurement. But E. coli Nissle 1917 is our final chassis, which expression ability is about 10 times lower than BL21 according to the eGFP fluoresence detection. And that's why we used J23100 to drive the expression of YgfU & pucL in our project in the end.
To conclude, according the relative capacity of promoters we determined, we selected appropriate promoter for the enzymes YgfU, pucL and 188.8.131.52. J23100 for YgfU & pucL, J23113 for 184.108.40.206.
 C. Lou, B. Stanton, Y.-J. Chen, B. Munsky, C. A. Voigt, Ribozyme-based insu lator parts buffer synthetic circuits from genetic context. Nat. Biotechnol. 30, 1137 (2012). doi:10.1038/nbt.2401 pmid:23034349
 2016 William & Mary iGEM Team, http://2016.igem.org/Team:William_and_Mary/RiboJ, 2017/10/01.