"
Team:Glasgow/LOVresults
From 2011.igem.org
Revision as of 23:02, 21 September 2011 by EmmaCampbell (Talk | contribs)
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. 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 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.
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.
| 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.
Figure 3: Image showing our LOV2 containing DH5alpha plated on IPTG and Ampicillan plate.
Figure 2: 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
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.
