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Team:RMIT Australia/Notebook
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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. | 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. | ||
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| + | 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. | ||
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| + | accccaggc''tttaca''ctttatgcttccggctcg''tatgt''tgtgtggaatt | ||
===30th June=== | ===30th June=== | ||
Revision as of 05:39, 2 July 2010
The Story So Far
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.
accccaggctttacactttatgcttccggctcgtatgttgtgtggaatt
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.
