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Parts Submitted to the Registry

An important aspect of the iGEM competition is the use and creation of standard biological parts. Each team will make new parts during iGEM and will place them in the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created . The "groupparts" tag will generate a table with all of the parts that your team adds to your team sandbox. Note that if you want to document a part you need to document it on the Registry, not on your team wiki.
Remember that the goal of proper part documentation is to describe and define a part such that it can be used without a need to refer to the primary literature. The next iGEM team should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.

Primers and PCR

When we realised that it would be quicker to try to extract genes via PCR rather than via a DNA library, we BLAST searched for genes present in the Tod operon, and found the whole operon was remarkably conserved, with only a couple of the genes needing any kind of degeneracy in their primers. We concentrated on genes within the operon that didn't have any restriction sites incompatible with the basic Biobrick standard:

TodX: (544bp)
>TODXF
5'-atgcccgccagtctgacgcttg-3'
>TODXR
5'-accagccagcaccatgcggc-3'
TodX gene in all putida the same regardless of strain

Photo of the gel to be added tomorrow
TodX - 544bp
> Aeruginosa - None
> Maxwell reaction
> Putida A and B - Bands at <500bp, 600bp, 1200bp and >1500bp
> Orange bacteria - 1 band at >1500bp
> Yellow bacteria - Faint bands at 1200bp and >1500bp
> Boilate reaction
> Putida A and B - Faint bands at <500bp, 600bp, 1200bp and >1500bp
> Orange and yellow boilates failed.

TodF: (460bp)
>TODFF
5'-atgggtgccgttggcgtgag-3'
>TODFR
5'-gtttttgcgatcagtcctccgcg-3'
All putida appear to have the above sequences,though other species may react better to:
>TODFR
5'-gtttttgMgatcagtcctccgYg-3'

photo to be added tomorrow
> TodF - 460bp
> Aeruginosa - None
> Maxwell reaction
> Putida A and B - 1 band between 700bp-800bp
> Orange bacteria - 1 band at 1500bp and 1 band between 1200-1500bp
> Yellow bacteria - Faint band between 500bp-600bp, other bands are above
> Boilate reaction
> Putida A and B - Faint band between 500bp-600bp, other bands are above, 1 strong band between 700bp-800bp
> Orange - Strong bands between 1000bp-1500bp
> Yellow - Faint band between 500bp-600bp, and strong bands >1200bp

TodC1: (1353bp)
>TODC1F
5'-atgaatcagaccgacacatcacctatc-3'
>TODC1R
5'-tcagcgtgtcgccttcagcg-3'
one strain has a C rather than a G base
>TODC1R
5'-tcaScgtgtcgccttcagcg-3'

photo to be added of gel
TodC1 - 1353bp
> Aeruginosa - band at 800bp and 1kb
> Maxwell reaction
> Putida A and b - Band at 800bp, 1.2kb and one in-between 1.2 and 1.5kb
> Orange bacteria - one band high up above the 1.5kb marker
> yellow bacteria - no bands
> Boilate reaction
> boilates failed to amplify

TobB: (324bp)
>TOBBF
5'-atgacttggacatacatattgcggcag-3'
>TOBBR
5'-tcacttcaactccccgttgtcgag-3'
^ all blast search results for this gene yielded 100% similarity

photo to be added tomorrow

TodB - 324bp
> Aeruginosa - None
> Maxwell reaction
> Putida A and B - None
> Orange bacteria - several non distinct bands ranging from >300bp to 800bp, 2 are between 300bp-400bp
> Yellow bacteria - several non distinct bands ranging from >200bp to 1500bp, 1 faint band is between 300bp-400bp
> Boilate reaction
> Putida A and B - No distinct bands
> Orange - one faint band between 300bp-400bp and more faint bands between 400bp-1000bp
> Yellow - None
TobG:(807bp)
>TOBGF
5'-atgagcgaactagataccgcgcg-3'
>TOBGR
5'-ttatgcctttgcaaaagcggcggtc-3'
^ all putida results had the same gene sequence

Photo to be added of gel tomorrow
TodG - 807bp
> Aeruginosa - band at 1kb and one high up
> Maxwell reaction
> putida A and b - band at 700bp and a fainter one at 800 and 600 ... bands also present above the 1.5kb marker
> Orange good sized band at 800bp and a lower one at 700bp ... also has bands at 1.2 and 1.5 kb
> yellow faint band at 800bp and one at 1kb
> Boilate reaction
> boilate failed

Unfortunately, with the 2 strains of P. putida, none of the primers amplified the target genes, meaning that the strains do not encode any of the Tod operon genes. Because of this, it meant we couldn't make a biobrick. However, these primers will be available for future iGEM Leicester teams, who with the right strains of bacteria, may be able to extract the target genes relatively quickly meaning that it could be easier to make a biobrick for them.

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