Difference between revisions of "Part:BBa K3285000"

Latest revision as of 00:50, 22 October 2019

Blue Light Repressible Promoter

This composite promoter consists of 6 basic parts that include 2 promoters, 3 coding sequences and one double terminator(Fig.1)

USAGE AND BIOLOGY

Fig.2: The working of the promoter in the absence of blue light

As we can see in Fig.2, there is a constitutive promoter that constitutively expresses the yf1 and fixJ genes. YF1 is a fusion protein made of YtvA (Light sensitive protein) and FixL (phosphorylates FixJ). The phosphorylated FixJ induces pFixK2 promoter and hence mrfp is expressed. As a result the RFP is constitutively expressed when there is no blue light (Fig.2)

Now, in the presence of blue light, the conformation of Ytv1 changes hindering the phosphorylation of FixJ by FixL leading to a reduced expression of mrfp gene. Increasing the intensity of blue light further hinders this interaction leading to an even lesser expression of mrfp gene. As a result, with increasing intensity of blue light, RFP production reduces.

Fig.3: The working of the promoter in the presence of blue light

We want to use this promoter to regulate the level of mutD5 (codes for the faulty epsilon subunit of DNA polymerase III of bacteria) in the cell thereby resulting in different levels of mutation rates in the bacteria. Our project aims at developing a tool for attaining tunability of mutation rates in E.coli using an external physical inducer (such as blue light) which will help in performing directed evolution faster and more easily.

CONSTRUCTION

Restriction free cloning (PCRs) was used for obtaining the final composite promoter and all
other intermediate parts which will be described below. First, Dterm, pFixK2 and mrfp were
ampilfied from their respective plasmids. For amplification of parts from plasmids, 50-100 ng
of template was used. For Dterm, annealing temperature (A.T) was 53 deg., for pFixK2 the
A.T was 58 deg. The extension period (E.P) for both was 18 sec. For mrfp, A.T was 54 deg.
and an E.P of 50 sec was used (Fig.4) The resulting bright bands obtained in each case were gel extracted and purified.

Constitutive promoter (C.P) and the YF1+FixJ construct were amplified from their respective plasmids. Here too, 50-100 ng of template was taken for amplification. For C.P, annealing temperature used was 55 degrees with an extension period of 15 sec. The band of interest was gel extracted and purified (Fig.5). For the YF1+FixJ construct on the other hand,

annealing temperature of 54 degrees and an E.P of 2 min was used which gave 2 bands one of which was the band of interest. This band was gel extracted and purified (Fig.6)

The first ones to be put together were Dterm and pFixK2 (TP). The two genes were used as template and a 3-step overlap extension PCR along with primers in a pot reaction was performed to stitch them together at an A.T of 52.2 deg. and an E.P of 30 sec. About 50 ng of pFixK2 was used and equimolar quantity of Dterm was used. Resultant product was gel extracted and purified (Fig.7).

Now the YF1+FixJ gene construct along with TP were used as templates for Overlap extension PCR for putting them together to make YTP. 31.7 ng of YF1+FixJ construct and equimolar concentration of TP was used. Here, a two-step PCR was used along

with its forward and reverse primers instead of a 3-step because the annealing temperatures of the primers were very close to the extension temperature of 68 deg. of the polymerase. An E.P of 2 min 20 sec was used. The obtained product was run on a gel and extracted (Fig.8).

Now, pFixK2 and mrfp were used as templates to perform extension PCR to make PR. 200 ng of mrfp and equimolar concentration of pFixK2 was used. First, a 3-step PCR with just the templates (without forward and reverse primers) at an A.T of 51 deg. And an E.P of 50 sec for 15 cycles. Now, the forward and reverse primers were added to the PCR mix and the A.T was kept the same whereas the E.P was changed to 3 min for another 15 cycles. The resulting product was gel extracted and purified (Fig.9).

Finally, YTP and PR were used as templates to perform an overlap extension PCR to obtain the final construct (CYTPR) using 215 ng of YTP and equimolar concentration of PR. This was done by first performing a 2 step PCR with just the templates (no primers) as the annealing temperature was higher than the extension temperature (68 deg.) of the polymerase (Because now the entire pFixK2 was an overlapping region). This was done for 15 cycles with an E.P of 2 min.

After 15 cycles, forward and reverse primers were added, of which, the forward primer consisted of the entire constitutive promoter. An A.T of 54 deg. And E.P of 3 min 10 sec was used. The product was run on a gel, gel extracted and purified. This product was now used as a template for re-amplification of the final construct. About 50 ng of template was used along with a completely new set of bigger forward and reverse primers. Since these primers are long and have an annealing temperature of 69-70 deg., again a two-step PCR was performed with an extension period of 3 min 10 sec. The resulting product was gel extracted and purified. Fig 10 shows that the concentration of the final construct has increased, since now, a much brighter band is obtained with just 2 uL of product.

MODELLING

Based on data provided in Möglich, Moffat and Ayers[1], a deterministic model was designed to explain the dynamics of the system.

As we can see, the maturation of rfp takes some time but in about 300 minutes, we are able to spot the difference between the rfp redout at different intensities of blue light. In the above figure, the thickness of the line represents the blue light intensity, the thicker the line, the more the intensity. Naturally, we see that with increasing intensity of blue light, rfp readout reduces.

In the above figure (Fig.2),we see the direct relationship between rfp readout and blue light intensity. With increasing intensity of blue light (y axis), RFP production (x axis) reduces very steeply. We hope to see similar results experimentally as well.

EXPERIMENTAL CHARACTERIZATION

For the characterization of this promoter, cultures were grown in LB under 3 different conditions- Dark(No light), white light and blue light. The growth in blue light was carried out using a “blue light box”. The assay was conducted in DH5alpha cells. 2 incubators were used:- one for dark condition and the other for white light and blue light condition. A dark environment was created by wrapping the falcons with aluminium foil and taping it to ensure it doesn’t have any opening for light to pass through. We took cells that constitutively express rfp, which was the cells containing the plasmid of constitutive promoter as our positive control and made 3 inoculations of them. The cultures were allowed to grow for 12 hours straight in the given conditions. After that, cells were pelleted, washed and resuspended in 1x PBS buffer. The solutions were normalised to OD600 value 1 and Fluorescence intensity was measured (Excitation: 584nm; Emission: 607nm). The results were as follows:-

As we can see incubating under blue light led to high repression of mRFP expression. The mRFP expression was easily observed in dark and white light. The very high mRFP readout under white light maybe due to unknown interactions with other wavelengths. Further characterisation may be able to show such interactions and explain this result better. As of now, the part as expected has repression of gene expression under blue light.

SEQUENCE AND FEATURES