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Team:UNAM-Genomics Mexico/Notebook/hydaopt
From 2011.igem.org
Optimization of HydA
CAI optimization and 5'UTR ΔGibbs optimization control
1 - General Overview
1.1 – Overview
One of the main aims in Synthetic Biology is the design and construction of new biological functions and systems not found in nature. One can accomplish this by assembling a new genetic system from scratch by adding elements (genes) from different species and introducing it into a new host. Each organism has evolve in a manner that permits the differential usage of codons to a specific set of genes; that is, constitutively expressed genes have their own usage of tRNAs, and so on. If an exogenous sequence is to be expressed in a host, and this happens to use some rare codons that have been reserved to other endogenous sequences, the host might result compromised in its ability to properly translate this endogenous sequences and so, its growth might get affected. It is of our concern not to affect the host of our genetic circuit, Rhizobium etli, that's way we've developed bioinformatic tools that allow us to modify the genes that form our system in a manner that they have the codons used by the constitutively expressed genes of R. etli; by doing this, we expect not to harm the bacteria by expressing our system.
We also modified the 5'UTR and a small portion of the coding region in the amino terminus that minimizes the probability of forming secondary structures that hinder the ribosome from binding the transcript and start the translation.
As all of our codifying region of our system has been optimized for CAI and for 5'UTR ΔGibbs, we expect that by comparing wild-type sequences against our optimized, to observe a growth difference between the Rhizobium etli carrying either of these variants.
1.2 – Control Components
The optimization control is composed of the following parts:
pBBR1MCS-5 derived plasmid. This plasmid is a modification of the pBBR1MCS-5 plasmid, which is a broad-host-range (bhr) vector designed to assist genetic anaylsis in prokaryotes, specifically for Gram- bacteria that are naturally CmR (chloramphenicol resistant). It is used to transfer genes from E. coli to R. etli by first transforming E. coli S17 competent cells with this vector containing an RFP reporter and later performing a conjugation between the transformed E. coli cells and R. etli CFN42.
Bba_J04500. This biopart will permit constitutive expression of the wild-type and the optimized sequences to be tested. This part is composed of a promoter regulated by LacI and a strong RBS. As the host were this part will be found does not contain the LacI repressor, a constitutive expression is therefore expected.
Bba_B0015. This biopart is a double transcriptional terminator consisting of BBa_B0010 and BBa_B0012. It has a high forward efficieny, it will ensure that the growth phenotype that could be observed in the host is due exclusively to the translation of the coding region and not to the excessive transcription from the plasmid pBBR1MCS-5.
HydA wild-type CDS. This sequence belongs to the hydrogenase I gene from Clostridium acetobutylicum ATCC 824. It is 1749 nucleotides long. HydA optimized CDS. This sequence was made by the bioinformatic tools we developed that change all the codons in the sequence by the most used codons by the constitutive expressed genes in Rhizobium etli CFN42. It is 1749 nucleotide long.
Two constructions are constructed, each composed of the vector pBBR1MCS-5, the part Bba_J04500, the part Bba_B0015 and differing in the coding region each one possess, either the wild-type or the optimized hydrogenase I, HydA gene.
