Difference between revisions of "Part:BBa K3198005"

Revision as of 05:14, 15 October 2019

Improved Blue light-repressible system with RFP reporter attached with YbaQ degradation tag

Team iGEM18 NUS Singapore-A submitted BBa_K2819103 which is a Blue light-repressible system with RFP reporter attached with YbaQ degradation tag.

Sequence and Features

Description

This part contains a promoter that can be repressed by blue light (450nm). This promoter makes use of a blue light dependent DNA-binding protein, EL222. Irradiation by blue light of wavelength 450nm exposes the hitherto sequestered HTH, facilitating dimerization of EL222 and subsequent DNA binding. The repression is achieved by placing the DNA binding site of EL222 between the -35 and -10 hexamers of the consensus promoter in Escherichia coli , creating the blue light repressible promoter PBLrep. As a result, EL222 acts as a repressor, blocking the binding of RNA polymerase and repress gene expression in the presence of blue light. In the dark, RNA polymerase can now bind, and gene expression takes place.

While many teams have previously characterized blue-light repressible systems, there are not many investigations done on the potential effect of spacer DNA behind the promoter on protein expression. The part BBa K2819103 by iGEM18_NUS Singapore-A had an 11 base pair long spacer DNA behind the blue-light repressible promoter. This year, we constructed two different new plasmids. One of them had an increased spacer DNA sequence with 30 base pairs, while the other one had the spacer sequence removed completely. Our aim was to investigate how the length of this spacer DNA would affect the performance of the blue-light repressible system.

Usage

This part contains a promoter that can be repressed by blue light (450nm). This promoter makes use of a blue light dependent DNA-binding protein, EL222. Irradiation by blue light of wavelength 450nm exposes the hitherto sequestered HTH, facilitating dimerization of EL222 and subsequent DNA binding. The repression is achieved by placing the DNA binding site of EL222 between the -35 and -10 hexamers of the consensus promoter in Escherichia coli , creating the blue light repressible promoter PBLrep. As a result, EL222 acts as a repressor, blocking the binding of RNA polymerase and repress gene expression in the presence of blue light. In the dark, RNA polymerase can now bind, and gene expression takes place.

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.

Improvement over existing part BBa_K2819103 by iGEM18 NUS Singapore-A

While many teams have previously characterized blue-light repressible systems, there are not many investigations done on the potential effect of spacer DNA behind the promoter on protein expression. The part BBa K2819103 by iGEM18_NUS Singapore-A had an 11 base pair long spacer DNA behind the blue-light repressible promoter. This year, we constructed two different new plasmids. One of them had an increased spacer DNA sequence with 30 base pairs, while the other one had the spacer sequence removed completely. Our aim was to investigate how the length of this spacer DNA would affect the performance of the blue-light repressible system. Our hypothesis is adding a longer spacer sequence would increase the RFP expression while removing the spacer sequence would decrease the RFP expression.

Characterization

The three types of plasmid were transformed into Escherichia coli MG1655 strain for characterization. For simplicity, the three types of cells are referred to as 0bp, 11bp and 30bp. 50uL of overnight culture of 0bp 11bp and 30 bp MG1655 cells were transferred into 5mL LB+Chloramphenicol medium in three 50mL tubes. They were then refreshed to a starting culture and incubated in 37 °C for one and a half hours until OD₆₀₀ reaches about 0.8 to start characterization.

12 well plates were used for characterization. The plate layouts are the same for both plates. Plate layout: first column: triplicate of 0bp MG1655, second column: triplicate of 11bp MG1655, third column: triplicate of 30bp MG1655, last column, triplicate of blank LB medium (Figure 1).


Figure 1: Plate layout for characterization of improved part

1mL of each cell and medium culture were transferred into each well in the 12 well plates. Initial OD₆₀₀ and RFP readings were taken at 0h time point using H1 Synergy microplate reader. The protocol is shaking for 10s, reading OD₆₀₀, and reading RFP. The results are exported as excel. For subsequent readings, the protocol is the same throughout and all results are exported in the excel sheet. After the first reading, 1 plate was placed on the blue light device to be exposed to blue light, while the other plate is covered with a black cloth to prevent any exposure to light (Figure 2). Both are incubated in a shaking incubator at 37 °C and shaking at a speed of 125rpm. Hourly readings were performed for the next 8h.


Figure 2. experimental setup for blue light and dark conditions

Results

The following figures (3 and 4) are the growth curves of cells with and without blue light exposure.


Figure 3. Growth curves of MG1655 transformed with plasmid containing blue-light repressible promoter and different length of spacer DNA under blue light environment

Figure 4. Growth curves of MG1655 transformed with plasmid containing blue-light repressible promoter and different length of spacer DNA under dark condition.

Generally, there is no significant difference between the OD₆₀₀ curves of cells in the dark and blue light condition. However, we noticed that under both Dark and Blue light conditions, the cells with a plasmid containing 30 base pair spacer DNA sequence grew faster and reached a higher final OD₆₀₀ as compared to 11bp and 0bp.

The following figures (5 and 6) illustrates the production of RFP in the dark and under exposure to blue light.



Figure 5. RFP production curves of MG1655 transformed with plasmid containing blue-light repressible promoter and different length of spacer DNA under blue light environment.



Figure 6. RFP production curves of MG1655 transformed with plasmid containing blue-light repressible promoter and different length of spacer DNA under dark condition.

We observed that the blue-light repressible system was working well. For cells incubated in the blue light environment, we could see that their RFP production decreased significantly over time while the cells incubated in the dark had an increasing RFP production. It is clear that cells containing the plasmid with 30 base pair spacer DNA had the highest RFP production overtime. The RFP production of this new construct is 3.8 fold as compared to the original construct (11 base pair). However, the cells with their spacer DNA completely removed (0 base pair) did not seem to produce RFP at all. Therefore, we can conclude that increasing the spacer DNA length behind the blue-light repressible promoter increases the RFP production while decreasing the spacer DNA reduces RFP production.

Combining the findings for OD₆₀₀ and RFP, we had an interesting observation: The cells producing higher amount of RFP (longer spacer length) also grew faster and reached a higher final OD₆₀₀. This is different from what we expected. We assumed that cells producing higher amount of RFP would have a greater metabolic burden and therefore grow slower.

The following figures (7 and 8) illustrate the ratio of RFP to OD₆₀₀.



Figure 7. RFP production per OD₆₀₀ curves of MG1655 transformed with plasmid containing blue-light repressible promoter and different length of spacer DNA under blue light environment.



Figure 8. RFP production per OD₆₀₀ curves of MG1655 transformed with plasmid containing blue-light repressible promoter and different length of spacer DNA under dark condition.

Taking into account of cell density, we also plotted the RFP/OD₆₀₀ curve. Our conclusion is clearly supported by these curves as well, with a longer spacer DNA sequence demonstrating a much larger fold change between production and repression of proteins. This therefore indicates that a larger spacer sequence would result in a more effective blue-light repressible system.

References

Takakado, A., Nakasone, Y., & Terazima, M. (2017). Photoinduced dimerization of a photosensory DNA-binding protein EL222 and its LOV domain. Physical Chemistry Chemical Physics, 19(36), 24855-24865.

Source

BBa_K2819103 originated from Erythrobacter litoralis obtained from iGEM18_NUS_Singapore-A

Design Considerations

Generally, the basal level of protein production under blue-light repressible promoter is low. In order to better characterize the promoter and clearly show the repression, we wanted to achieve higher basal protein production. We hypothesized that a longer spacer sequence between the promoter and coding region might allow easier binding of the transcription factors and faster formation of transcription initiation complex which might lead to higher protein production. We also carefully designed the sequence of the spacer DNA such that it does not interfere with other parts of the gene, especially the coding sequence.