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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.
Perform restriction digest to ensure we have LOV2 domain.

Transform LOV2 domain into DH5alpha

Make glycerol stocks of LOV2 containing DH5alpha

Design primers for LOV2 containing biobrick ends and PCR up LOV2 domain.

Design site directed mutagenesis primers to get rid of illegal pst1 site

Perform site directed mutagenesis on LOV2

Make LOV2 fluoresce under UV light.

Ligate LOV2 into sumbission vector

Submit LOV 2

Methods

LOV2 domain was transformed into DH5alpha cells using standard protocol. Overnights were made, and then glycerol stocks.

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

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

The sequence for LOV2 contains an illegal pst1 site, therefore we performed one round of site-directed mutagenesis on it using the forward and reverse SDM primers shown in Table 1.

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

Streaked LOV2 containing DH5alpha from glycerol stocks onto 1mM IPTG and Ampicillan agar plates. Kept in dark for 3 hours at 30 degrees. Used Blak Ray lamp, and transilluminator on plates to visualise any fluorescence. No fluorescence was seen.


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

Made up liquid cultures of DH5alpha + LOV2 in 2ml of LB + 2 microlitres Ampicillan. Liquid cultures grown for 4 hours and then 0.5 ml plated onto plates with varying concentrations of IPTG and with 1mM Ampicillan. These were then wrapped in tinfoil and allowed to grow in the 30 degree incubator for 24 hours. They were tested for fluorescence using both the Blak Ray lamp and the transilluminator with riboflavin as a positive control. No fluorescence was seen.

Liquid cultures were grown, then transferred to varying concentrations of IPTG, kept in darkness and incubated at 30 degrees for 3 hours. These were then illumuinated using the transilluminator and the Blak Ray lamp to test for fluorescence. Riboflaving was used as a positive control, and ready grown Top 10 cells without the LOV2 domain were used as a negative control. No illumination was seen of the LOV2 domain.

Figure 3: Image showing liquid culture of DH5alpha cells grown with IPTG compared to positive control riboflavin for fluorescence.

Illumination was tested by spinning down overnight cells containing LOV2 into a pellet and transilluminating with riboflavin as a positive control. No fluorescence was 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 4: 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