Team:UPIBI-Mexico/Project

From 2012.igem.org

(Difference between revisions)

Latest revision as of 17:12, 18 July 2012

Constitutive chimeric promoters for the expression of recombinant proteins

Ever wanted to produce huge amount of your favorite recombinant protein? Here we will show you how!

In this part of the project we aim to develop constitutive chimeric promoters that could allow for accumulation of different levels of recombinant proteins in Chlamydomonas reinhardtii chloroplast. The psbA promoter is one of the strongest promoters in green eukaryotic algae. This promoter has been used in the past (reference) to drive the expression of foreign proteins. Unfortunately, this promoter seems to render high level of protein accumulation only when the endogenous psbA product, the D1 protein, is absent. This has led some people to think that the D1 is somehow regulating the transcription of the ORF under the control of the psbA promoter. We think that since the psbA mRNA levels are fairly constant throughout the growing cycle, the D1 protein is actually regulating translation of the mRNA by interacting with the 5´UTR. We reasonedº that if we can relieve the mRNA from this control element, but keeping its transcription at high level, a higher level of recombinant protein accumulation could be achieved. We think that replacing the psbA 5´UTR with other 5´UTR that have been shown to work well in prokaryotes can do this. Given the similarities of the chloroplast with prokaryotes, the effectiveness of these 5´UTRs should remain to a certain extent.

Regulated expression of recombinant proteins

Tired of those easy to degrade yet expensive proteins? Try our regulate expression Chlamy system to turn on the expression only when you want it.

In this part of the project we want to develop a device that could allow for the regulated expression of recombinant proteins. We are particularly interested in this part because we think that if we can switch on the expression of a protein once a culture has reached the stationary phase or shortly before it, the accumulation rate of the recombinant proteins could be higher that the degradation rate and thus result in a higher yield. This application could result particularly attractive for proteins whose turnover rate is high and its accumulation results difficult. There is no previous report on the use of the lacI/Plac in Chlamydomonas chloroplast so in this part of the project we will try to use it to drive the expression of mCherry protein.

Regulated expression of enzymes contained in an operon

Imagine an illuminated or colorful world with algae that emit light or produce bright red carotenoids. Follow the light here!

This part of the project we want to build and characterize a device that could allow for the regulated expression of an operon containing a set of enzymes. We have chosen the araC/PBAD circuit to regulate the expression of the lux operon developed by the Cambridge iGEM team in 2010. Alternatively, the regulation of a series of enzymes involved in a metabolic pathway could be used to turn on the pathway only once the stationary phase has been reached. This could be particularly important for secondary metabolites such as carotenoids and vitamins.

@@ Line 14: / Line 14: @@
 <center><h3>Ever wanted to produce huge amount of your favorite recombinant protein? Here we will show you how!</center></h3>
-<div style="text-align:justify">In this part of the project we aim to develop constitutive chimeric promoters that could allow for accumulation of different levels of recombinant proteins in Chlamydomonas reinhardtii chloroplast. The psbA promoter is one of the strongest promoters in green eukaryotic algae. This promoter has been used in the past (reference) to drive the expression of foreign proteins. Unfortunately, this promoter seems to render high level of protein accumulation only when the endogenous psbA product, the D1 protein, is absent. This has led some people to think that the D1 is somehow regulating the transcription of the ORF under the control of the psbA promoter. We think that since the psbA mRNA levels are fairly constant throughout the growing cycle, the D1 protein is actually regulating translation of the mRNA by interacting with the 5´UTR. We reasonedº that if we can relieve the mRNA from this control element, but keeping its transcription at high level, a higher level of recombinant protein accumulation could be achieved. We think that replacing the psbA 5´UTR with other 5´UTR that have been shown to work well in prokaryotes can do this. Given the similarities of the chloroplast with prokaryotes, the effectiveness of these 5´UTRs should remain to a certain extent.
+<div style="text-align:justify"><font color="white">In this part of the project we aim to develop constitutive chimeric promoters that could allow for accumulation of different levels of recombinant proteins in Chlamydomonas reinhardtii chloroplast. The psbA promoter is one of the strongest promoters in green eukaryotic algae. This promoter has been used in the past (reference) to drive the expression of foreign proteins. Unfortunately, this promoter seems to render high level of protein accumulation only when the endogenous psbA product, the D1 protein, is absent. This has led some people to think that the D1 is somehow regulating the transcription of the ORF under the control of the psbA promoter. We think that since the psbA mRNA levels are fairly constant throughout the growing cycle, the D1 protein is actually regulating translation of the mRNA by interacting with the 5´UTR. We reasonedº that if we can relieve the mRNA from this control element, but keeping its transcription at high level, a higher level of recombinant protein accumulation could be achieved. We think that replacing the psbA 5´UTR with other 5´UTR that have been shown to work well in prokaryotes can do this. Given the similarities of the chloroplast with prokaryotes, the effectiveness of these 5´UTRs should remain to a certain extent.</font>
 </div>
 </div>