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From 2011.igem.org

Results for the light responsive constructs

Light is a ubiquitous feature of nature; it is a fundamental aspect of life on Earth, from driving photosynthesis in plants to allowing sight in humans. It is also an indispensable tool in the engineering of biological pathways.

The DISColi project aims to capitalize on light in the control of circuits which allow the modular construction of valuable products. In contrast to other potential response-inducers, such as temperature or chemicals, light has the ability to be finely adjusted. The wide-ranging spectrum of wavelengths which constitute light offer innumerable possibilities to the user; with a simple and inexpensive set-up including a light-source and a filter, one is capable of fine tuning which wavelength is used. In tandem with a variety of different light-dependent devices, this system allows for precise control over what the desired outcome will be. Light can also yield high resolution results, meaning an enhanced formation of 3D structures and bio-photolithographic systems.

We aimed to build a series of constructs of OmpC, OmpF and Bluf promoters, connected to eYFP (BBa_I13001) and a constructed RFP reporter, to determine whether the light responsive promoters work at all, and eventually characterise them.

This work would serve as a proof of concept that expression of our novel biobricks via light is feasible.

Materials and Methods:

List of the biobrick used:

- B0030, Strong RBS, Ap+

- B1006, Strong double terminator, Ap+/Kn+

- E1010, RFP, Kn+

- I13002, Weak RBS – eYFP – double terminator (sYFP), Ap+

- I13001, Strong RBS – eYFP – double terminator (wYFP), Ap+

- K238013, Bluf promoter, Ap+

- R0084, OmpF promoter, Ap+

- R0082, OmpC promoter, Ap+

- K225000, GlrN (green light receptor), Cm+

And the biobricks received from the Edinburgh team (all in pSB1C3, Cm+):

- EDIN 1, lac promoter with phycocyanobilin biosynthesis pathway - based on BBa_K322122

- EDIN 2, corrected cph8 + RBS without promoter - based on BBa_K322124

- EDIN 4, cph8 + pOmpC-eYFP - based on BBa_K322126

Cells

- Top10 chemically competent cells from Invitrogen

- Δ EnvZ CP919 E.Coli cells (BBa_V1012)

Top10 cells were initially transformed with each of the biobricks, overnighted and miniprepped.

CP919 cells were made competent and stored until ready to be transformed with the completed constructs.

RFP assembly method:

The RFP biobrick was digested with EcoRI-SpeI and ligated to the strong double terminator, previously cut with EcoRI-XbaI. The obtained ligation was then inserted into Top10 cells and selected on agar/kanamycin plates.

At the same time a library of Promoters/RBS was made.

(check here our toolbox of promoter-RBS!)

The RFP-Terminator construct was then digested with XbaI-PstI and ligated to each of the available Promoter/RBS, after previous digestion with SpeI-PstI.

The obtained Promoter-RBS-RFP-Terminator constructs were then digested with EcoRI-PstI and ligated into the submission vector.

Edinburgh biobricks:

We attempted to ligate EDIN1 plus EDIN2, and EDIN1 plus EDIN4, in order to obtain a functional light receptor together with the full phycocyanobilin pathway. The successfull ligations were then transformed into ΔEnvZ cells containing either Bluf-YFP, Bluf-RFP, OmpF-YFP, OmpF-RFP, OmpC-YFP or OmpC-RFP. The resulting double transformants were then selected on agar/chloramphenicol/carbenicillin plates.

Results:

In the first place we wanted to test the reliability of our constructs and we wanted to see with our own eyes to what extent is gene expression affected by a weak or strong ribosome binding site.

We then grew the cell cultures containing all the component to detect light, in dark and light and compared the fluorescence to determine the functionality of the light responsive system.

The image above shows how the strength of the RBS influences the levels of expression in Top10 E.coli cells. These cells are lacking the machinery required to detect red light (phycocyanobilin path. + cph8) therefore the expression at the OmpF promoter is constitutive. The intensity of the YFP is reflected by the strength of the RBS.

The gel electrophoriesis of the two version of the OmpF-YFP construct show that the cells we used for microscopy contained the correctly assembled construct.

This set of pictures above show incomplete light responsive constructs.

Unfortunately we realised too late that the EDIN1+2 ligation was in fact either just an EDIN1 which reannealed on itself or a dimerised plasmid backbone, byproduct of the ligation reaction.

The controls show a constitutively expressed RFP under control of an OmpF promoter lacking the light sensing machinery.

The negative control shows the green haze at the air-culture border under the coverslip. We suggested that this type of green fluorescence is due to the LB media in which the cells were cultured.

The series of images shown here provide unconsistent results: - any RFP under control of OmpC is supposed to be transcriptionally silent or very weakly expressed. The RFP fluorescence could be due to an RFP on the backbone of the GlrN vector, but we’re unable to check that on the registry. - the OmpF-RFP containing culture contained also a wrong EDIN1+2 ligation thus the red fluorescence was expressed in a constitutive manner. We are anyway unable to show that the fluorescence was due to a correctly assembled construct.

Positive results for Bluf-sYFP

The Bluf-sYFP grown in light (5th picture from the top) shows higher YFP expression levels than the same culture grown in darkness (6th from the top). The gel electrophoriesis on the left handside shows the right band pattern for Bluf-sYFP after digestion with EcoRI-PstI, proving that this behaviour was due to the presence of a correct construct. On the other hand the results for OmpF-sYFP grown in light and dark are swapped around and both samples contain a non-functional red light receptor. The increase in fluorescence can be simply due to an higher density of cells in the dark-grown sample or by an advanced state of growth (lumps of cells are visible).

The OmpC samples glow of a faint red regardless of the absence on any RFP in the construct. again, we can speculate about the presence of such an RFP on the backbone of the GlrN vector.

Discussion

The experiment worked as expected only for the Bluf-sYFP construct, and unfortunately we didn’t manage to produce a Bluf-RFP construct to corroborate our result.

The OmpF and OmpC promoters didn’t behave as expected for a number of reasons:

- the cells didn’t contain all the machinery required to detect light. GlrN also required the presence of the phycocyanobilin chromophore to work so we’re not surprised that OmpC wasn’t responsive to light.

- the phycocyanobilin pathway present in cells containing OmpF-reporter construct lacked the cph8 receptor.

- All the ligations of OmpC /reporter construct didn’t produce a positive result. The reason why we failed to ligate the two was probably due to the large amounts of DNA that went lost when extracting the digested insert from its agarose gel, and our failure to balance the amounts of insert and backbone to be used in the ligation reaction.