Team:NUS Singapore-A/Improve
Overview
We have improved the existing part BBa_K2332021, which was submitted by iGEM17_UCL, using our new part, BBa_K2819103. We hoped to understand the dynamic characteristics of the blue light repressible promoter (PBLrep) better as it has a pivotal role in our project. The existing part has a bioreporter, the coding region of the bioreporter, LuxCDABE. However, the large gene cluster (>5500bp) makes it difficult to carry out dynamic characterization due to its complexity. Furthermore, no characterisation data is available on iGEM17_UCL’s part registry page for the blue light repressible promoter BBa_K2332019.
Therefore, our team sought to conduct a study on PBLrep by using a more suitable reporter. We altered the sequence of this composite part by replacing the LuxCDABE coding region with a commonly used reporter gene, the red fluorescent protein (RFP, ~700bp), as well as attaching different degradation tags to the reporter gene to study the characteristics of this promoter. Furthermore, this new part will have wider applications compared to the previous part, such as by serving as an indicator. As the overall plasmid size is reduced significantly, it is now easier for users to co-transform it with other constructs.
Our new part: Blue Light Activated Repressible Promoter with RFP Reporter attached to YbaQ Degradation Tag
This part contains a promoter that can be repressed by blue light (450 nm). The promoter makes use of a blue light dependent DNA-binding protein, EL222. Irradiation by blue light exposes the hitherto sequestered HTH, facilitating dimerization of EL222 and subsequent DNA binding. Repression is achieved by placing the DNA binding site of EL222 between the -35 and -10 hexamers of the consensus promoter in E. coli, creating the blue light repressible promoter PBLrep As a result, EL222 acts as a repressor, blocking the binding of RNA polymerase and repressing gene expression in the presence of blue light. In the dark, monomeric EL222 is unable to attach to its binding site, and RNA polymerase can now bind to drive gene expression (Figure 1).
Biology
Originating from the marine bacterium Erythrobacter litoralis HTCC2594, EL222 is a photosensitive DNA binding protein, with a N-terminal light-oxygen-voltage (LOV) domain and a C-terminal helix-turn-helix (HTH) DNA binding domain.
Characterization of New Part
To characterize this promoter, we used a common reporter gene, RFP (red fluorescence protein). However, we quickly found that the expression of RFP cannot be regulated by this promoter, as its expression profile did not follow induction patterns. Therefore, we altered the sequence of the coding region to obtain a degradation rate optimal for this promoter to work. This improve its functionality as users can now a have better knowledge on how to use this promoter. To achieve this, we attached different degradation tags to RFP, and performed characterizations using various blue light on-off patterns. PBLrep can be regarded as a constitutive promoter when blue light is absent, i.e. in the dark, as dimerization of EL222 will not take place.
All plasmids confer kanamycin resistance, and all constructions and characterizations were done in E. coli TOP10. Three composite parts were used: PBLrep-RFP (original part), PBLrep-RFP-DAS and PBLrep-RFP-YbaQ. All three were tested simultaneously in each experiment. Engineered cells were inoculated in 5 mL of LB with kanamycin overnight. The next day, 5 uL of cell culture was refreshed in 5 mL of fresh LB with kanamycin until OD600 reaches 0.05, upon which 1 ml of cell culture was transferred to each well of a 12-well plate. Cells were then incubated in 37°C at 120 rpm under different light conditions depending on the experimental setup. Fluorescence of the cells was measured at 1-hour intervals using BioTek Synergy H1 microplate reader.
Cells were characterised in 12-well-plates in this experiment, and each sample was measured in triplicates. An example of a setup is shown below.
‘Blk’: blank (LB + kanamycin), ‘Ctrl’: PBLrep-RFP, ‘DAS’: PBLrep-RFP with the DAS degradation tag, ‘YbaQ’: PBLrep-RFP with the YbaQ degradation tag
Characterizing under Blue Light
In this experiment, one setup was incubated under persistent blue light for 8 hours, while another setup was incubated in the dark (covered with black cloth) for the same duration.
Our data suggests that using RFP solely as a reporter is unable to reflect the expected induction and repression activities of the promoter. As seen from Figures 3a and b, fluorescence decreased over the period of 0 to 4 hours in the dark, and increased over the period of 4 to 8 hours under blue light. This is completely opposite of what we expected. We speculated that this phenomenon is due to the mismatch in the rate of increase in OD600 and the degradation rate of RFP. Hence, to alter the degradation rate of RFP, we attached 2 different degradation tags to it, YbaQ and DAS. Degradation tags are also referred to as degrons. They are either part of coding regions, or are added to them to direct their degradation by proteases.
Experimental results show that RFP attached to YbaQ could reflect the expected induction and repression activities of PBLrep. As shown in Figure 3a, RFP/OD increased steadily with time in the dark, where repression is absent, while in Figure 3b, we can see that RFP/OD decreased exponentially with time under blue light. The high intial RFP/OD at 0 hour in Figure 3a can be explained by the low initial OD600 (0.05) and the presence of RFP in the system prior to the commencement of the experiment.
[Formula: RFP/OD_light off ÷ RFP/OD_light on], numbers on top of bar graphs represent the fold change.
Figures 4a and b represent the comparison of RFP/OD of persistent light on and off conditions for PBLrep-RFP and PBLrep-RFP-YbaQ. There is a clear increasing trend for the fold change for PBLrep-RFP-YbaQ, while the fold change for PBLrep-RFP increased over the period of 0 to 4 hours, then dropped for the subsequent 4 hours.
This characterisation experiment tells us that the degradation rate of RFP-YbaQ is required for this promoter to work as intended.
To verify that the degradation rate of RFP-YbaQ is indeed the most optimal for PBLrep to work, we carried out further characterisation experiments with different blue light on-off patterns.
As observed from the Figures 5 and 6, even under different light on-off patterns, PBLrep-RFP-YbaQ still performed the best, showing clearer oscillation patterns with the absence and presence of light, while PBLrep with just RFP alone or with RFP-DAS showed relatively poor responses with the rate at which light is being switched on and off.
We also modelled the activity of the promoter and our model shows that for PBLrep to reflect better oscillation patterns, we should incubate the cell cultures in the dark for 45 minutes, and under blue light for the remaining duration of the experiment.
Our wetlab experimental data verified the recommendations made by our model. A dip is no longer observed at the start of the graph, and the change in RFP/OD could reflect our light on-off pattern.
Conclusion
Our improvement of changing the coding region sequence of a common reporter gene, RFP, by adding a degradation tag, YbaQ, has allowed us to better understand how we can use this the blue light repressible promoter PBLrep. Based on our characterization results, we recommend future users to use a coding region with similar degradation rates as RFP-YbaQ in order to achieve the desired function of this promoter. This new part has hence improved the functionality of this promoter, which has helped us to be more mindful when we select genes to be placed under the control of this promoter in our project.
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