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From 2010.igem.org

The Story So Far

16th July

The project outline has been put online. Symon has begun outlining the human practices side of the project.

2nd July

Primers for Quikchange mutagenesis were designed to turn the -35 and -10 element into restriction sites.

-35

• F 5' tcattaggcaccccaggccttaagctttatgcttccggctcg 3'
• R 5' cgagccggaagcataaagcttaaggcctggggtgcctaatga 3'

-10

• F 5' gctttatgcttccggctcggatcctgtgtggaattgtgagcgg 3'
• R 5' ccgctcacaattccacacaggatccgagccggaagcataaagc 3'

1st July

Designing the protein expression vector. The two characteristics that we are looking for are:

• Strong promoter
• Inducible

In order to achieve these two aims, we will use the biobrick BBa_R0010 which is an (lac) inducible promoter and incorporate a T7 promoter to it. This process involves the removal of the -35 and -10 promoter sites and the incorporation of the T7 promoter sequence.

By mutagenesis we aim to turn the promoter elements into restriction sites (which are not found in the plamsid), thus being able to remove the promoter section and then introducing the T7 promoter sequence by ligation.

5' ACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATT 3'

In bold are the -35 and -10 promoter sites (respectively). The -35 element can be mutated into the AflII restriction site (c.ttaag), while the -10 element will be mutated into the BamH1 restriction site (g.gatcc).

30th June

We found the T.aquaticus DNA polymerase sequence from the NCBI website. From this, we designed primers that contained restriction site "sticky ends" on the 5' and 3'. The restriction sites that are going to be used for introducing Taq into the vector are XbaI (5') and SpeI (3').

Forward primer:

5' CTA GAA TGA GGG GGA TGC TGC CCC TCT TTG AG 3'

Length: 32
GC Content: 56.3 %
Tm: 65.6 ºC

Reverse Primer:

5' GAT CAT CAC TCC TTG GCG GAG AGC CAG TCC 3'

Length: 30
GC Content: 60.0 %
Tm: 66.2 ºC

Due to the high GC content of the Taq gene, the Tm of the primer is very high. A two step PCR reaction might be needed.

29th June

The team is starting to design the experiment. We need to:

• Design primers to introduce Taq into psB1C3
• Create a good protein expression vector.

22th June

Work has begun on creating primers to extract the protein we are working on from its wild-type plasmid into a plasmid which conforms to biobrick cloning standards.

19th June

Jeremy Nagels, a student at Monash university, Ebony and Symon have begun discussing various project ideas. These include:

Oscillators

The use of the Hydrogenase 3 complex to generate Hydrogen within E Coli as a form of biofuel.

The possibility of biological prevention of tooth decay with biobricks.

The application of biobricks to a mixed cell community.

The development of cell free transcription/ translation within a cellular system, for example the phloem of a plant, (although the development of this level of symbiosis may be the stuff of future projects)

The attempt is being made to develop a project that in some ways reflects these interests and possibly taps a common thread underlying them.