Promoters and terminators are very important devices in the engineering design of synthetic biology. They are located upstream and downstream of ORF, respectively, controlling the transcription initiation and termination. Thus, both elements control the net protein expression of synthetic constructs.
Previous studies have shown that for optimal protein expression, stronger terminators are recommended so that aberrant transcripts can be avoided. Therefore, our team aims to characterize a potentially strong terminator,BBa_K3320008, to benefit the iGEM community.
The arabinose-induced GFP expression plasmid—pGR-ECK120015170—which contains araC operon, araBAD promoter, RBS, GFP, ECK120015170 terminator and RFP (Figure 1.) from Ying-Ja Chen’s work was used to characterize terminator ECK120015170. The GFP gene was set upstream and RFP was placed downstream of the ECK120015170 terminator.
Figure 1. The design of the main part of genetic circuit
The terminator strength is further generated by using the equation
The subscript Term refers to measurements in the presence of terminators, and the subscript 0 refers to measurements in the control group. Here the control lacks the terminator sequence, so transcription does not stop between GFP and RFP. TE refers to terminator efficiency.
Previous studies have shown the feasibility of using green fluorescent protein (GFP) as a quantifiable reporter gene, so it was available to use the intensity of GFP fluorescence to reflect the amount of protein expression upstream. Also, red fluorescent protein (RFP) has shown to be available to reflect the efficiency of protein expression. Therefore,the strength of the terminator was reflected by the ratio of the expression level of two fluorescent proteins: GFP and RFP. Ying-Ja Chen’s study showed that the average GFP intensity under the treatment of arabinose is 15714.00, and the average RFP intensity under the treatment of arabinose is 141.53. Without the induction by arabinose, the GFP intensity is 62.90 and the RFP intensity is 14.50. (Figure 2.)
Moreover, the predicted terminator strength (Ts) is 99.59. Previous study shows that Ts above 10 is corresponding to terminator efficiencies greater than 90% . 99.59 is much larger than 10, so terminator ECK120015170 is a very strong terminator.
Figure 2. Fluorescence from GFP and RFP are measured in the uninduced and induced states
The detection of terminator strength is important in the process of protein expression. During literature review, we discovered a novel and easy assay to detect the strength of terminators. By calculating the ratio of upstream GFP fluorescence intensity to downstream RFP fluorescence intensity, the terminator strength can be generated. This method of detecting terminator intensity will be a great help to other teams in the iGEM community, and in the future they can also easily use this assay to detect the intensity of terminators in their projects.
Limitation of the experiment
Although we found a good detection assay for the terminator during our literature review, we did not perform this assay because of the limitation of laboratory equipment, so we referred to previous experiments and cited their results. In the future, if the conditions are available, our team as well as other iGEM teams will continue to carry out this method.
The promoter we characterized is called araBAD promoter. araBAD promoter is found in bacteria and especially as part of plasmids used in laboratory studies. araBAD is the gene originally downstream of this promoter, and the repressed protein of this promoter is araC. We experimentally characterized this promoter,BBa_K3519010, because araBAD is a common promoter that may be present in studies by other iGEM teams, so we wanted to add some data. In our study, we used arabinose to induce the GFP expression and investigated the araBAD promoter.
Figure 3. The Snapgene gene mapping of the araBAD promoter
Our experiment utilized the arabinose-induced GFP expression plasmid to characterize arabinose-induced promotor. The arabinose-induced GFP expression plasmid—pGR-ECK120015170—contains araC operon, araBAD promoter, RBS, GFP, ECK120015170 terminator and RFP. (Figure 4.)
Figure 4. The design of the main part of genetic circuit
We titrated the induction of arabinose by varying the concentration of the inducer. The ratio of GFP relative intensity to OD value was the amount of protein expression despite the effect of strain concentration. The amount of protein expression was corresponding to the efficiency of the promoter.
Previous studies have shown the feasibility of using green fluorescent protein (GFP) as a quantifiable reporter gene, so it was available to use the intensity of GFP fluorescence to reflect the amount of protein expression upstream. The GFP intensity over OD value can show the amount of protein expression. The ratio due to the different concentration of arabinose is visualized in Figure 5.
Strength of the experiment
In this study, we characterized the araBAD promoter and contributed to the iGEM community. The amount of protein expression was probed by setting a concentration gradient of arabinose and observing the changes in GFP fluorescence intensity. By analyzing the experimental data, we found that the value of GFP/OD showed a logarithmic growth image as the concentration gradient of arabinose increased.
Limitation of the experiment
When the inducer arabinose was added, the way in which arabinose enters the cytosol was not taken into account. Unlike other inducers, arabinose requires a transporter protein called araE to assist its entry into the cytosol. Therefore, probably because this was not taken into consideration, our gradient induction results were not rigorous enough. Also, according to the graph in Figure 5, the GFP/OD value does not reach the upper limit when the arabinose concentration is increasing.
Last year, our team (BNDS_China 2021) used the rhamnose induction system to induce antibiotic resistance genes as part of the directed evolution system. Although team iGEM14_Wageningen_UR had characterized the rhamnose operon previously, Data about its steady state analysis, optimal inducer concentration, and leakage are still insufficient. This year, part of our project focused on the efficiency of induction systems. Thus we plan to fill the lacking data of the rhamnose operator using kinetics. By finding the steady state (the proteins' generation and degradation at the same velocity) of the rhamnose-induced protein expression, we are able to directly relate the protein expression level to the concentration of rhamnose. Data are also documented on BBa_K1493501
We build a rhamnose-induced GFP expression plasmid for characterization. The steady-state could be found by looking for a stage that GFP fluorescence/ABS 600 is stable.
8 Groups of different RHA concentrations: 0mM, 0.1mM, 0.5mM, 1mM, 2.5mM, 5mM, 7.5mM, 10mM, 20mM each with 6 repetitions were tested with proper controls (blank, WT strain, and constitutive GFP expression strain) in the same assay. Abs 600 and GFP fluorescence were read every 10 minutes, and the total duration of the program was 24 hours.
Figure 6. The value of fluorescent level/Abs600 in different concentrations of rhamnose in their steady state.
By finding the steady state of strains in each group of concentration, we build a standard curve (fig.1) of different concentrations of RHA induction. With higher rhamnose concentration, the Abs 600 of the bacteria culture will be higher when reaching a steady state. The critical point of rhamnose's concentration is 10mM. At 10 mM, the GFP fluorescence/Abs 600 in the steady state reaches the maxim value; after 10mM, there is no significant growth in GFP fluorescence/Abs 600 as the concentration of Rha increases.
Figure 7. The value of fluorescent level/Abs600 in different concentrations of rhamnose in their steady state.
We use the data to compare the efficiency of the CV system and Rha system. Compared to the Rha system, the CV system has higher cost-effectiveness and less leakage when we perform the expression.
In the Rha system, the expression leakage is nearly 2.5 times its last concentration. In comparison, the leakage of CV expression is only about 1.5 times its last concentration. The precision of expression is a significant advantage.
We compare the official price of CV and Rha powder on Solarbio. The powder of crystal violet is 60 RMB for 25 grams, and the powder of Rhamnose is 210 RMB for 5 grams. For each trial, we did the same concentration and identical numbers of sets; the cost of Rha is about 300000 fold higher than CV's when reaching the maximum protein expression level.
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[2] Chen, Y.-J., Liu, P., Nielsen, A. A., Brophy, J. A., Clancy, K., Peterson, T., & Voigt, C. A. (2013). Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nature Methods, 10(7), 659–664. https://doi.org/10.1111/tpj.14907
[3] Cao, H., & Kuipers, O. P. (2018). Influence of global gene regulatory networks on single cell heterogeneity of green fluorescent protein production in bacillus subtilis. Microbial Cell Factories, 17(1). https://doi.org/10.1186/s12934-018-0985-9
