Edinburgh/Yoghurt/Wet Lab

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Revision as of 14:05, 3 October 2007

Introduction | Applications | Design | Modelling | Wet Lab | References

Biobrick Creation

Overview of the process we used to generate our biobricks:

1. Designed primers, which included a ribosome binding site and the biobrick restriction sites, EcoRI, XbaI, PstI and SpeI, to amplify our desired gene out of its host organism

2. Purified the PCR product and loaded onto an agrose gel to check for the presence of PCR product of the right size

3. If a band was present at the right size, we digested the PCR product and vector with the restriction enzymes EcoRI & PstI

4. Digested PCR product and vector were ligated overnight with T4 DNA ligase

5. Ligated vector and gene were then transformed into E. coli and plated onto Blue/ White selection Amplicillin plates

6. Transformants that contained a product ligated into the vector would grow as white colonies and are easy to select

7. Minipreps were prepared of the white colonies

8. Purified vector DNA was digested with restriction enzymes EcoRI and PstI to determine if the vector insert was of the correct size

9. Vectors containing inserts of the correct size were sent for sequencing to confirm if they did indeed contain the correct gene

The complete methods we used to generate our biobricks may be found on the [http://openwetware.org/wiki/French_Lab French Lab] OpenWetWare site

Zeaxanthin Synthesis Pathway

Biobricks created so far

• CrtE
• CrtBI (with PstI restriction sites)
• CrtZ

Primers

Vanillin Biosynthesis Pathway

Biobricks created so far

• Sam8

Genes sent to GENEART for synthesis

• COMT
• ech

Primers

Multi Host Plasmid pTG262

The plasmid we recieved had a multicloning site containing EcoRI, XbaI and PstI sites. To convert pTG262 into a biobrick vector we inserted a biobrick between the EcoRI and PstI sites, this simultaneously removed the intravening XbaI site, and introduced all four biobrick restriction enzymes sites (EcoRI, XbaI, SpeI & PstI).

To create the biobrick restriction sites, we inserted a total of three biobricks:

• Plac-RFP (gives red transformants)
• Plac-lacZ (gives blue transformants)
• Ptet-RFP

In order to test the plasmid in a multitude of gram negative bacteria, including Shewanella, Pseudomonas & Agrobacterium, we will transform the pTG262-Plac/RFP construct into our chosen host. This will enable easy detection, as all bacteria capable of retaining and expressing the vector will form easily detected red colonies, when grown on media containing lactose.

Introduction | Applications | Design | Modelling | Wet Lab | References