Team:Hong Kong-CUHK/PROJECT RESULTS

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RESULTS

I. Biobrick Construction Strategy
The Sensory Rhodopsin system works in E.coli by fusing SR and Htr with a flexible linker and joint the HtrII membrane-proximal cytoplasmic fragment with the cytoplasmic domain (Methyl-accepting chemotaxis protein (MCP) signalling domain) of the eubacterial chemotaxis receptor.[1] The fusion protein in BBa_K317028 and BBa_K317003 used the Tar gene from Synechocystis.typhimurium, have their construct design based on [2]. The previous study made three different junction constructs (P, G, M) based on studies of dimerization, yet no accurate tool was used for domain determination. Therefore, we made the following improvements.

1.         Tar gene from E.coli K12 strain was used instead of that of S.typhimurium, as we believe E.coli can express and function more properly with its native genes and proteins.
2.         We designed the construct by using an accurate protein domain determining tool [3], the whole E.coli Tar protein consist of three domains- Tar ligand binding domain (amino acid sequence 1-175), the HAMP domain (amino acid sequence 194-263) and the Methyl-accepting chemotaxis protein (MCP) signalling domain (amino acid sequence 264-553). As only the cytoplasmic domain is needed, the exact Methyl-accepting chemotaxis protein (MCP) signalling domain (amino acid sequence 264-553 of EcTar) was cut and fused with membrane-proximal cytoplasmic fragment part of HtrII (amino acid sequence 1- 133).
3.   Restriction sites of HindIII and BamHI were added before and after the SRII gene respectively for enabling switching the sensory rhodopsin portion of the fusion protein. According to previous study. [2] A series of mutant sensory rhodopsins have been identified which covers a large variation of absorbing spectrum. These two restriction sites allow further switching of the sensing unit, so the light sensing system can be tuned for sensing different kinds of light source.
Further integration of other peptides (e.g.: His-tag), for the construction of a larger fusion protein is also possible. (HindIII for N-terminal while, BamHI for C-terminal)

The improvements made were successful as the function (sense light for cell movement) of the above fusion proteins were tested with positive results shown below.

II. Method of measurement
According to previous studies on positive phototactic microorganisms, colonies of microorganisms should spread towards the light source. [4] 

As our cells receive stimulation of blue light from all directions instead of unidirectional as what the paper used, therefore, they spread out in all directions after 12 hr exposure of light. Average diameters were measured by a electronic ruler with precision = ± 0.01 mm. Data of diameters from at least three independent clones were collected. Paired t-test was used to analyze the collected data. A significant difference was observed between the plates (*** indicates p < 0.001).

1. Main Page – Promoter efficiency for BBa_K786001. BBa_K786002, BBa_K786003

To test the expression of sensory rhodopsin triggered by constitutive promoter BBa_J23100 to sense light,

we need to test the effect conferred by different E. coli strains to expression of red fluorescence protein reporter downstream of BBa_J23100 to different bacterial strains. It allows us to select the suitable strain(s) for this constitutive promoter for expressing sensory rhodopsin.

Florescence plate reader was used to take readings of fluorescence emission of 635nm and absorbance at 600nm (OD600) between time intervals of 12 hours on each strain. The measurements were started when the cultures reached a OD600 of around 0.4 that represents log phase of active proliferation. Growth curve and fluorescence intensity against time were plotted to compare cell growth and protein expression on different strains.

Three independent experiments were conducted. No significant difference was observed on the growth curves, indicating a similar growth rate among the three bacterial strains with BBa_J23100 transformed. It implies the promoter does not cause cell toxicity or growth inhibition of these three bacterial strains.

For the protein expression, the results showed that the fluorescence intensity of reporter in DH5α was significantly lower compared with TOP10 and BL21(DE3).

To conclude, DH5α is not an optimal strain to utilize promoter BBa_J23100, while TOP10 and BL 21(DE3) can effectively express the reporter. Therefore, in downstream application of our light sensing biobricks (BBa_K786001, BBa_K786002, BBa_K786003) in which BBa_J23100 was used, DH5α are not used.

2. Main Page – Positive Phototactic Construct for Blue Light Detection (BBa_K786002)

To evaluate whether our biobrick BBa_K786002 causes cell movement under blue light exposure, we transferred transformed bacteria on soft agar and observe if it moves under blue light. It is known that bacteria can swim on soft agar.

Soft agar (0.4%) plate was prepared. Cell cultures transformed with BBa_K786002 (6, 12 and 25 µl) with OD600 ~2 were pipetted on each semi-agar plate. Duplicate aliquots were done on each plate and four plates were made. Two of the plates were placed in dark and two were placed under blue LED light of 200 mW (with spectra covering from 430-480 nm).

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