Difference between revisions of "Part:BBa K2332001"

Latest revision as of 10:46, 25 October 2017

Blue light inducible expression system with GFP reporter (Pblind GFP)

This composite part consists of a blue-light inducible promoter (Pblind) upstream the gene encoding green fluorescence (GFP) codon optimized for E. coli. For blue-light transcriptional induction, cells must also express EL222 (BBa_K2332004). As shown in Figure 1, Pblind 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, a natural photosensitive DNA-binding protein from the marine bacterium Erythrobacter litoralis HTCC2594. 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. In darkness, EL222 will reverse to its repressed state spontaneously. This optogenetic tool allows a dynamic, rapid and switchable transcriptional control. This biobrick allowed us to test the effectiveness of the Pblind promoter by measuring the basal transcription of GFP in the absence of the blue light transcriptional activator EL222.

Figure 1: Blue light inducible expression system. Under blue light, the EL222 DNA binding protein dimerises and bind 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

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.

Sequence and Features