Difference between revisions of "Part:BBa K2332002"

Latest revision as of 01:40, 22 October 2021

Blue light inducible promoter (Pblind)

Promoter activated by EL222 (BBa_K2332004) upon blue-light exposure (465nm)

Pblind is a blue-light inducible promoter that will only allow RNAP to transcribe genes downstream upon blue-light (465) exposure, as this will induce a conformational change in EL222, a natural photosensitive DNA-binding protein, required for RNAP recruitment and transcriptional activation. To enable blue-light dependent transcriptional control, EL222 should be constitutively expressed by the cells.

Since light can be controlled easily in space, time and degree, this new basic part will enable tight spatiotemporal control of gene expression.

Usage and Biology

This part consists of a fusion of the EL222 DNA binding region and the LuxI promoter. The lux box, a 20bp inverted repeat (LuxR and 3-oxo-C6-HSL complex binding region) from the luxI promoter, was replaced with the 18bp DNA binding region of EL222. In the dark, EL222 is inactive as its N-terminal LOV domain represses its DNA-binding C-terminal HTH domain. Upon blue light exposure (450nm), LOV-HTH interaction is released, allowing it to dimerize and bind its binding region, overlapping the -35 region of the luxI promoter. This ultimately results in the recruitment of RNAP and transcriptional activation.

Figure 1: Blue light inducible expression system. Under blue light (465nm), the EL222 DNA binding protein dimerises and binds its binding region within the designed Pblind promoter, overlapping the -35 region of the luxI promoter. This ultimately results in the recruitment of RNAP and transcriptional activation. Figure adapted from Jayaraman P. et al. (2016)

Characterisation: Experimental setup

10-beta E. coli cells were transformed with GFP constructs under the control of different promoters: J23151-GFP (positive control), R0040-GFP (negative control), Pblind-GFP or not transformed (WT). 23151 is a constitutive promoter, R0040 is a TetR repressible promoter (repression inhibited only by the addition of tetracycline), Pblind promoter is a fusion of EL222 (photosensitive transcription factor) binding region and the luxI promoter, where EL222 is only able to dimerize and bind the Pblind promoter upon blue light exposure, where it can then recruit RNAP and drive the transcription of genes downstream.

Colony PCR was used to identify successful transformants. For each construct, 3 of the identified colonies were aliquoted into 5ml of growth media (LB media with 25ug/ml Chloromphenicol), and grown overnight at 37°C in darkness (covered in aluminium foil) or exposed to blue light (465nm). Each biological replicate was then diluted in LB to OD600=0.6. 200μLx4 of each biological replicate were aliquoted into a black flat bottom 96 well plate. LB media with 25ug/ml Chloromphenicol was also aliquoted for fluorescence baseline determination. The fluorescence of all repeats along with the LB was measured using a Tecan Safire 2 Multi-Mode Plate Reader.

Results

Figure 2: Blue light inducible promoter (Pblind) characterisation. Wild type (WT) 10beta cells were transformed with J23151-GFP (positive control), R0040-GFP (negative control) or Pblind-GFP. J23151 is a constitutive promoter, R0040 is a TetR repressible promoter and Pblind promoter is a fusion of EL222 binding region and the luxI promoter. All cells were either grown in the dark or under blue light overnight. GFP fluorescence was measured using a Tecan Safire 2 Multi-Mode Plate Reader. Error bars represent the SD of 4 technical repeats of 3 biological replicates per condition. The statistical significance of **** P < 0.0001 was calculated using the Tukey's multiple comparisons test.

Analysis

This construct allowed us to test whether the promoter Pblind has any significant leakage. We wanted to show that GFP cannot be expressed in the absence of EL222. This is of particular interest as the aim of LIT is to demonstrate the versatility and high precision of light control. As shown in Figure 2, only J23151-GFP (positive control) had a significant difference in fluorescence compared to R0040-GFP (negative control), Pblind-GFP, WT cells and the Luria Broth (LB) in both dark and Blue-light conditions. Pblind-GFP had no significantly different fluorescence level compared to the LB baseline, negative control or WT cells in either condition. This is expected, as the EL222 protein is required for blue-light inducible transcriptional activation.

Characterization from XMU-China 2021

Group: iGEM Team XMU-China 2020

Characterization

In some designs of pBLind-EL222 system, EL222 was usually expressed by a constitutive promoter in the Anderson family, such as BBa_J23119 and BBa_J23106. Although the EL222 protein is activated to bind pBLind promoter due to the conformational change upon blue light illumination, the binding events still happen by chance in dark environment, which results in unexpected transcriptional leakage of the genes controlled by pBLind promoter. Reducing the existent pool of EL222 protein in the cell will low down the leakage level in the dark state. Therefore, using an inducible promoter to control the expression of EL222 seems to be an ideal option to lower the leakage when the blue-light irradiation is not supplied.

Fig. 1.The illustration and characterization of the system. (A)Gene circuit illustration for the system.(B) RFUsfGFP/OD₆₀₀ of the system in dark and blue-light condition was calculated as time progressed.

The result showed that the system maintained a well blue-light sensitivity and a higher expression level of sfGFP in the system was observed as time progressed (Fig. 1B). It demonstrated that the EL222 was able to work as a photosensitive DNA-binding protein and pBLind could be activated by EL222.

Sequence and Features