"

From 2011.igem.org

iLOV Results

Aims

iLOV (K660003) is an optimised version of the LOV2 fluorescent domain (K660000). It shows better fluorescence and photostability. We aim to format this with biobrick ends no illegal restriction sites, and ligate it into the submission vector in order to make it into a fluorescent reporter. The activity of iLOV can be monitored by it's fluorescence.

Methods

Due to the number of illegal restriction sites within iLOV, we had the construct synthesised and codon optimised for expression in E.coli.

Evolutionary, organisms have a bias towards certain codons. Due to the degenerate nature of the genetic code, there is more than one codon present for most amino acids. E.coli an organism with well studied codon preferences, and for this reason, we had the iLOV constuct synthesised in such a way that it was codon optimised.(Sharp et al., 1988)

Once the construct had been synthesised we ligated it into the submission vector and submitted it to the registry.

Results

The iLOV domain was successfully synthesised and submitted into the submission vector.

iLOV shows fluorescence under an excitation wavelength of 476nm, with an emission of 510-550nm.

Figure 1: Gel electrophoriesis of iLOV Biobrick (labeled b) and iLOV construct (labeled c). the samples are alternated uncut and EcorI-PstI double digest. each iLOV variant was cloned onto the pSB1C3 submission vector before digestion. each second sample from the ladder shows two clear bands: the submission vector at 2kb and the iLOV insert at around 400kb. iLOV construct size: 404bp. iLOV biobrick size: 336 bp.

LOV2 Results

We have converted LOV2 into biobrick format by undertaking site-directed mutagenesis, adding biobrick ends and ligating it into a submission vector. (K660000)

Aims

LOV2 is a fluorescent domain which we have aimed to engineer as a reporter. Under excitation with the correct wavelength of light (476nm), fluorescence can be measured at an emission spectra of 510-550nm.


Figure 2: Graph showing absorption specra of LOV2 wild type (solid line) and a LOV 2 double mutant. Graph taken from:Christie, J.M. et al (2007) Steric interactions stabilize the signaling state of LOV2 domain of phototropin 1. Biochemistry, 46 pp. 9310-9319

We obtained the LOV2 domain contained within a PUC vector in Top 10 cells. To ensure that we indeed have the domain, we were required to do a restriction digest. We also transformed DH5alpha cells with the LOV2 domain and made glycerol stocks.

The main aim for us in order to obtain the LOV2 domain was to get it into a biobrick format without any illegal restriction sites. This involved us designing specific PCR primers which contain the biobrick ends, and using these to PCR up our LOV2. After this we were required design primers for, and to do site-directed mutagenesis to get rid of the illegal pst1 site contained within the LOV2 sequence.

In order to test that the LOV2 domain works, we aimed to test the fluorescence emisson.

Once LOV2 has been adapted to suit the biobrick format and has been tested for activity, we were required to ligate it into the submission vector and submit it to the registry.

Methods

Checking we have LOV2 -


Restriction digest was set up to ensure that we had LOV2 domain. Results show that we indeed had it.


Formatting LOV2 into a Biobrick:


The LOV2 domain does not contain biobrick ends in nature, therefore we specially designed the forward and reverse primers shown in Table 1. The LOV2 domain was then PCRed up using these primers to ensure the finished PCR product had the correct biobrick ends. This PCR product was the transformed into Top 10 so fluorescence could be tested.

One round of site-directed mutagenesis was performed on LOV2, using the forward and reverse SDM primers shown in Table 1. This got rid of the illegal pst1 site.

Table 1

Name of the primer	Sequence	Melting Temperature (oC)
LOV2 Forward	5'-GTGTGTGAATTCGCGGCCGCTTCTAGAGTCGCTGAAGGATCCAAGG-3'	73
LOV2 Reverse	5'-GTGTGTCTGCAGCGGCCGCTACTAGTATTATTAAACGTGGTCGGAACC-3'	72
LOV2 SDM Forward	5'-CGCAAAGGCGGTCTTCAGTACTTCATTGGTG-3'	64
LOV2 SDM Reverse	5'-CACCAATGAAGTACTGAAGACCGCCTTTGCG-3'	65

Testing LOV2 Fluorescence -


LOV2 transformed Top 10 was grown on IPTG and Ampicillan plates.

LOV2 was ligated into the submission vector. This product was then digested and run on a gel to ensure that it was successful.
.
The LOV2 which has been successfully ligated into the submission vector was submitted to the registry.

Results

Figure 6: Image showing e.coli with an empty vector (left) compared to those containing the LOV2 sequence when viewed with UV light. Image from : Christie, J.M. et al (2007) Steric interactions stabilize the signaling state of LOV2 domain of phototropin 1. Biochemistry, 46 pp. 9310-9319

References

Sharp et al, 1988. "Codon usage patterns in Escheria coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the considerable within-species diversity." Nucl. Acids Res.16, pp. 8207-8211