Team:Buenos Aires/Results/Bb2


Revision as of 01:18, 22 September 2012 by Abush84 (Talk | contribs)


Construct Desing based on Biobrick Registry Parts


His Secretion in Bacteria In the spirit of the competition we decided to design an extra construct besides our main biobrick, solely by using parts that came in the iGEM Kit 2012 distribution, and that could work for the same purposes that our main biobrick.

We designed a plausible construct; however we found several obstacles as we state here.

From this exercise we can conclude two things: - Some biobricks have not yet been optimized to be standard and simple to use in one step; it would only be desirable for them to be as easier to use as possible. - Our main biobrick is an important contribution to the registry part, given that it allows the export of aminoacids to be enhanced with the use of only one part, without the need of the many steps that we describe in this section.


To create a biobrick that would enhance Histidine secretion in E. coli using standard parts of the registry as a proof that one can increase the production and secretion of an aminoacid and its measurement in the culture medium. To learn how to use and merge standard parts from the registry provided at the iGEM Kit. To characterize the functioning of existent parts in the registry and new combinations of them, therefore contributing the improvement of the iGEM record.

Parts to use

Promoter (constitutive or inducible); RBS; Peptide Signal (Secretion Tag), Histitide Tag; Terminator.

Promoter choice

We found many usable parts to use as promoters and chose:

Promoter + RBS: BBa_K081005

Inducible Promoter (IPTG) + RBS (Strong): BBa_J04500

We would use the second one, so that the system is plausible of regulation.

Signal Peptide Options

Unfortunately, we found very few signal peptide biobrick options, solely two and tested for cyanobacterium.

"pilA1 signal sequence from cyanobacterium Synechocystis; secretes protein": BBa_K125300 (partially confirmed) tggctagtaattttaaattcaaactcctctctcaactctccaaaaaacgggcagaaggtggt

"slr2016 signal sequence from cyanobacterium Synechocystis; secretes protein" :  BBa_K125310 (partially confirmed) tggcagcaaaacaactatggaaaattttcaatcctagaccgatgaagggtgga

We could use any of them, but knowing that they are fit for Cyanobactierium, not E. Coli.

HisTag Options

Methionine + His Affinity Tag x 6: BBa_K133035 (partially confirmed)

Methionine is there only to be able to express the protein, so this is a HisTag, and it is functional to our purposes. We would put this 3 times in a row to make the histag stronger but then we have 2 questions:

- the methionine that remains in the middle of each histag, would it make the structure unstable?

For example, placing it three times in a row we get: atgcaccaccaccaccaccac/atgcaccaccaccaccaccac/atgcaccaccaccaccaccac

- This sequence does not carry a Stop Codon and we found that several of the parts of the registry do not carry one either, which could be a major issue.

His Tag BBa_K157011 (Bad Sequencing :( ) Discarded for Bad Sequencing

Stop Codon

There are no stop codons in the HIsTag availables at the Kit. Therefore we would need to use another biobrick carrying a stop codon and use its restriction sites to cut it and keep only the Stop Codon part.

There is a biobrick that has several things and then ends with a hisstop. It has several restriction sites before a 6His and Stop, so we could use this part as a biobrick if we were able to cut it.

In this site we can see the restriction sites present at this construct:

What restriction enzyme do we use in order for the remaining part to be used as a biobrick together with the other ones?


There are several options for terminators

Doble terminador: BBa_B0015 ; BBa_B0024

From Coliphage: BBa_B0012

Any of them would be ok for our purposes.