Regulatory

Part:BBa_K360041

Designed by: Jorge Eduardo Buendía Buendía   Group: iGEM10_UNAM-Genomics_Mexico   (2010-09-12)

Minimum Blue Light Receptor Promoter

This is a short version of the promoter BBa_K238013 activated by the blue light receptor system YcgF/YcgE, the original promoter was described and characterized by the K.U. Leuven 2009 team. This short promoter includes nucleotides from 26-75 of the original sequence reported by the K.U. Leuven 2009 team.

Originally, we tried to amplify the complete promoter region BBa_K238013 (86 bp) from genomic DNA of E. coli K12 by PCR but we failed in several trials. So we decided to synthesize the promoter in a primer and then insert it into pSB4A5 by PCR. Because of lenght limitations in primer synthesis we reduced the promoter region to 50 bp.

The reduced promoter retains the fundamental parts of the original one, these are the -35 and -10 box, the spacer between them, the inverted repeat 1 and 2 (inverted regions are the binding sites for the YcgE repressor) and the transcription start site. In order to test the functionality of our Minimum Blue Promoter we succesfully ligated it to our Strong RBS BBa_K360031 and the GFP BBa_E0040.

The YcgF/YcgE system is based on the action of the repressor YcgE, which is bound to the promoter region when there is no blue light, thus inhibiting the transcription of any gene downstream this promoter, as shown in the next figure.

No blue light.jpg

In the presence of blue light, YcgF dimerizes and now it has a great affinity for YcgE, clearing the promoter and allowing transcription to proceed.

Blue light jbuendia.jpg

It is reported that the response of this promoter is weak in comparison to some others standard strong promoters registered in the Registry of Standard Biological Parts. We implemented a protocol for testing the response of our Minimum Blue Light Receptor Promoter (Min-BP) in which we irradiated cells with the construction Min-BP + RBS BBa_K360031 + GFP BBa_E0040 with blue light (470 nm) leds for different times. We also irradiated with green (540 nm) and red (660 nm) light leds to discard any crosstalk of these wavelengths. GFP expression was compared to a reference: J23101 promoter + RBS BBa_K360031 + GFP BBa_E0040.

We measured GFP expression of our constructions, in order to irradiate the cells with Min- BP + RBS BBa_K360031 + GFP BBa_E0040 we used blue leds and irradiated the cells for 300 minutes, we also measured GFP expression of cells that were incubated in the dark and cells with the constuction J23101 promoter + RBS BBa_K360031 + GFP BBa_E0040.

For the experiment, cells were incubated overnight in M9 medium with glycerol (0.4%) as carbon source, in the morning OD600 was measured and cells diluted to 0.07 nm. After dilutions, we started irradiation with blue leds. Temperature is a very important factor in the function of the YcgF/YcgE system; it is reported that at 25ºC the ratio between the YcgE repressor and YcgF activator allows a good repression unless there is blue ligh irradiation, so we incubated cells with this promoter at 25ºC. Cells with the constitutive promoter BBa_J23101 were also incubated overnight at 37ºC.


A graph showing GFP expression of our Minimum Blue Promoter compared with a constitutive promoter is shown here:


Comparision with constitutive promoter.jpg


Next graph shows GFP expression in presence and absence ob blue light:

Blue light vs dark.jpg


We have shown that our Minimum Blue Light Receptor Promoter works as expected but the response is weak compared to a standard, constitutive promoter BBa_J23101. This results are consistent with previously reported data by the K.U. Leuven 2009 Team.

In order to increase GFP expression in response to blue light, an amplifier could be used. This means to ligate a T7 polymerase downstream the Minimum Blue Promoter and the GFP downstream a T7 promoter. Additionally, we can increment irradiation time and observe response of our blue light-induced promoter.


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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