Part:BBa_K786002:Experience

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Charaterisation of BBa_K786002

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. 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. 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).
We placed the plates overnight for 12 h at 25oC and compared the diameter differences between the plates with or without blue light exposure. We used paired t-test for analyzing the collected data. A significant difference was observed between the plates (*** indicates p < 0.001). The average diameters of three clones exposed under blue light are bigger than the counterparts in dark. Blue light triggers a change in diameter of 180 ± 40 %, while there is no significant change in diameter for those without BBa_K786002. When we put the bacteria transformed with BBa_K786002 under light, the blue light stimulates SRII and switches on the hisitine kinase CheY by decreasing the phosphorylation level of CheY (CheY-P). Therefore, a prolonged running period in bacteria is resulted as CheY-P causes cell tumbling, a random movement. When the cell tumbles and faces towards light, SRII is stimulated again. The process repeats and the cell will eventually travel towards blue light.

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Future Application

Together with Negative Phototactic Construct for Blue Light Detection (BBa_K786001), Phototactic Construct for Orange Light Detection BBa_K786003, Light-triggered Gene Expression System BBa_K786010 and the genome targeting CRISPR/Cas systems BBa_K786031. This part will have the following applications.

I. Light-directed bacterial cells sorter

When the phototactic systems are integrated with heavy metals collection devices [1, 2] , such as SmtA and MntH for collecting cadmium ions, fMT and Glpf for collecting arsenic ions, and BBa_K346005, a mercury(II) ions absorption device, our sensory rhodopsin systems could become a useful tool for heavy metal sorting in present in sewage.

When different ion-absorption systems are integrated with our Sensory Rhodopsin systems, different kinds of ions can be directed to different position for heavy metals sorting and separation in sewage, which could be a new tool for heavy metal recycling.

Metal ions collection aided by light-directed cell sorting in an environmental friendly way

Other than integrating with heavy metal absorption device, our sensory rhodopsin system can also be incorporated with our gene expression system and our genome targeting system in order to sort out and collect heavy metal ions while minimizing potential environmental hazards due to release of genetically engineered organisms.

First of all, the cells absorbing two different ions can be sorted out by blue light alone with positive phototactic device (BBa_K786002) and negative phototactic device (BBa_K786001).

An orange light source can then trigger the genome targeting system (BBa_K786031) together with the gene expression device for sensory rhodopsin (BBa_K786010).

The cells will eventually destroy their own genetically engineered DNA, and leading to cell death and releasing the heavy metal ions. Furthermore, this step can prevent horizontal gene transfer due to the disposal of cell debris to the environment, minimizing the environmental impact.

II. Improvement on the HR desalination system

Our sensory rhodopsins system (BBa_K786003) and the halorhodopsin system (BBa_K559010) are both sensitive to long wavelength visible light source. They can be integrated to enhance the desalination efficiency [3]. As E. coli can be attracted to a region closer to the light source with greater intensity, the halorhodopsin system should function with a higher efficiency and absorb more Cl- ions.

BBa_K786002 the phototactic device sensitive to near-UV light can be integrated to become a tool for cells/Cl- ions direction to a separate chamber, so that the remaining solution can achieve desalination.

III. Incorporation with BBa_K786031 as a new biosafety approach

Genetically-modified organisms have been put in use extensively throughout the last decade. Yet, concerns about such organisms causing undesirable effect to the environment once released have been raised. Our biobricks might be one of the answers to tackle such concerns. When the gene expression system for sensory rhodopsin is incorporated with the CRISPR/Cas system, bio-engineered cells would cleave their own DNA into nucleotides once exposed to natural light.
This system would be useful for bio-engineered cells that could be used to work in the dark. (e.g.: Biofuel synthesizing cells, human hormone-producing cells)

[1] PKU 2010 iGEM Team

[2] Tokyo-NoKoGen 2010 iGEM Team

[3] CUHK 2010 iGEM Team

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