Team:SCU-WestChina/Measurement

index

Meaurement
SCU-WestChina 2017 used insulator RiboJ and eGFP to measure the absolute protein expression ability of 10 promoters in Anderson's promoter family for the first time. After measuring the relationship between the absolute protein quantity and fluoresence with purified eGFP, we measured the eGFP fluoresence driven by different Anderson's promoters to achieve this goal. The measurement provides basic information for metabolic pathway construction because the proper absolute enzyme concentration is always needed to be consider to be consistent with the substrate concentration, which is never characterized in iGEM before.

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).

Fig.1 For eGFP purification, we chose His-tag as the proteintag and BL21(DE3) as the expression strain. After bacterial lysis, centrifugation and affinity chromatography we got eGFP shown on the figure. You can see the details of protein purification from the protocol.
Fig.2 Trying to make sure the accuracy of our standard curve, we decided to purify eGFP ulteriorly to enhance the purity of eGFP. So we chose to utilize molecular sieve chromatography and we got eGFP we wanted. What the peak of this figure shows is our eGFP during the process of molecular sieve chromatography.
Fig.3 SDS-PAGE was applied after the purified protein was sieved by molecular sieve chromatography. The 29kDa protein which had a high purity was enhanced green fluorescence protein, eGFP.

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.

Fig.4 The overall experiment result of Group A.

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.

Fig.5 Relative Capacity of Different Promotors (J00 short for J23100, J04 short for J231004…).

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 4.1.1.97. J23100 for YgfU & pucL, J23113 for 4.1.1.97.

Reference

[1] 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

[2] 2016 William & Mary iGEM Team, http://2016.igem.org/Team:William_and_Mary/RiboJ, 2017/10/01.

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