"

From 2011.igem.org

LOV2 Results

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.

We obtained the LOV2 domain contained within a PUC vector in Top 10 cells from.... 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 is 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, it is required that we ligate it into the submission vector and submit it o 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.

Figure 1: Image showing LOV2 PCR product run on gel

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 -

Figure 4: Image showing how e.coli should look 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

LOV2 containing DH5alpha cells were tested for fluorescence after growth on IPTG and Ampicillan, both liquid and plate forms and using both a Blak Ray lamp and a tansilluminator. Fluorescence could not be detected (Figures 2 and 3)/br>

Figure 2: Image showing LOV2 containing DH5alpha plated on IPTG and Ampicillan plate. No fluorescence can be seen.


Figure 3: Image showing liquid culture of DH5alpha cells grown with IPTG compared to positive control riboflavin for fluorescence. No fluorescence can be seen.

Why no illumination has been seen:
The LOV2 sequence and construct that we have been working with lacks a promotor, which is the reason fluorescence was not detected throughout the above experiments. Although fluorescence has not been detected, the LOV2 sequence we have been working from is the sequence of the LOV2 domain. We can therefore confirm that the biobrick part submitted is LOV2.
LOV2 was ligated into the submission vector. This product was then digested and run on a gel to ensure that it was successful.
Figure 5: Image of a gel showing successful ligation of LOV2 into submission vector.
The LOV2 which has been successfully ligated into the submission vector was submitted to the registry.

Results

iLOV Results

Aims

1) Design iLOV construct to be synthesised


2) Get iLOV synthesised


3)Transform iLOV cells and make fluoresce


4)Ligate iLOV into sybmission vector


5)Submit iLOV

Methods

iLOV was synthesised to get rid of illegal restriction sites. It was synthesised in such a way that it contains an RBS and a double terminator.


This synethesised product was then ligated into the sumbission vector and submitted to the registry.

Results