User:Jszab:m13:refactor

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M13.1 refactoring scheme.
In this refactoring scheme i will try to avoid changing the genes necessary for development. Stopping of one of those genes may suppress development. It might be necessary to temporarily stop the production, for example to release prepared phages when necessary.



Decoupling of genes
First thing to do before refactoring is selection of sequences and copying them in linear fashion. The genetically active elements are known and present in the registry of standard biological parts. They can be added to a piece of DNA using the parts.mit.edu website. Each of the genes were separated by the double of zero-cutters - unique restriction sites in the genome. This will allow for exchange of a given part.

Decoupling of functions
As I wrote at the beginning, I do not want to change the whole cycle. There are many genes which are produced one after another, or simulatenously. It will be technically difficult to control the life cycle properly even with strictly inducible promoters and terminators. Moreover, we would need more expertise to do it. Instead, I added the single OmpR promoter in front of Gene VII, which can be coupled to the light sensing mechanism described by Voigt [2005] http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html. This will allow to control easily the amount of output in a high throughput way: by shining the patterns and gradients on a dish with growing bacteria. The slice with plaques will show what should be the strength of the promoter. Thereby we will be able to tell what is the strength of production necessary, and backtrace which promoter could give this particular strength, given known condition of translation control. This will give a cue how the transcription of genes VII and IX takes place.

However, my main point is to make a phage useful for the protein display.
I would like to make a platform for useful genome changes of protein p8 as now it is impossible to add bigger residues to the protein and still obtain a viable phage. This paper comes particularly handy to solve this problem: http://chem.ps.uci.edu/~gweiss/SidhuS-2000-JMB.pdf

Another way, worth considering is addition of cysteine residues to p8 - innately. It will be necessary to add just a few CYS residues on the terminus which will allow for biotinylation and attachment of proteins produced outside of a phage.
Debugging

Copying of DNA from one place to another can yield having repeated functional sequences, such as RBSs or repeated promoters. To achieve efficiency of transcription it would be necessary to remove promoters, and removing RBSs is crucial to obtain functional protein sequences.
I did this task by alignement: first by selecting the promoter, or RBS to test, and then checking how similar are sequences around the genome. In order to perform this task efficiently some information about promoters and RBS of E.Coli was necessary:
This website contains information about the strength of the promoters and their structure:

http://www.mun.ca/biochem/courses/3107/Topics/promoter_bacterial.html

A figure exemplifying graphically RBSs:
rbs1055.logo.gif
Along with an article describing anatomy of RBS:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11601857&dopt=Abstract

The check list of refactoring
1) Put the genes apart by extracting the coding regions to separate biological parts and put them togather using parts.mit.edu
2) Add known promoters in front of the genes
3) Add two of the zero cutters enzymes in between the promoter RBS and genes end.
4) Change the distance between RBS and start codon of every ORF to the same length as in native genome.
5) Remove internal RBS sites and promoters to avoid repetition.
6) Add the features useful for engineering: for example OmpR promoter, in between of the unique sites so they can be easily exchanged.

BBa_M31111 - composite part of a part of a roughly refactored genome. Includes parts with nucleotides changed to deleted Promoters and RBSs.

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