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Team:MIT/Projects/Project1
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[[Image:HMX1_plasmid.png|center]] | [[Image:HMX1_plasmid.png|center]] | ||
| - | + | The gene that we decided to follow was the ''PcyA'' gene, from the ___ genome. After looking at the codon usage charts for baker's yeast, and realized that we would need to do codon optimization to more effectively produce this enzyme in baker's yeast. The codon usage comparisons of ''PcyA'' before and after codon optimization, as done by GeneART, is seen below. | |
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| + | [[Image:PcyA_Usage.png|center]] | ||
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| + | We synthesized this optimized ''PcyA'' using GeneART, and then cloned it into the plasmid, as shown below. | ||
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| + | [[Image:PcyA_plasmid.png|center]] | ||
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| + | With these two plasmids, we were planning to transform these into a yeast strain with the proper plasmids to be able to complete the functional assay. Unfortunately, there were a lot of problems in creating this functional strain | ||
Revision as of 04:06, 20 October 2009
Metabolic Engineering of PCB Synthesis in Yeast
As shown in the image below, phycocyanobilin (PCB) plays a crucial part in the PhyB-PIF3 system. Once PCB is in the active conformation it allows the phytochrome (PhyB) to bind to the integrating factor (PIF3). This image represents an experiment completed by the Quail lab at UC Berkeley, and is actually the basis of our functional assay.
PCB has two conformations, one which is active and the other which is inactive. PCB is initially in the inactive conformation and has the ability to absorb red light, at a wavelength of 680 nm, called the Pr conformation. Once PCB absorbs red light, it switches to the active conformation, called the Pfr conformation. In the Pfr conformation, PCB has the ability to absorb near infrared light, at a wavelength of 720 nm. Once PCB absorbs the near infrared light, it will switch back to the inactive conformation. This is detailed in the image below.
As was said before PCB plays a crucial part in the PhyB-PIF3 system. Without this chromophore it is impossible for this process to continue. We wanted to make this process self-sufficient. Therefore if the yeast was able to synthesize the PCB itself, it would not have to always be supplemented.
Developing the Standard: PCB from Spirulina
We decided to use a standard used in many other experiments involving phytochromes. Phycocyanobilin (PCB) extracted from Spirulina is a commonly used standard, as Spirulina produces a large amount of chromophores. We used Spirulina which was bought at Vitamin World as it is commonly used as a dietary supplement.
Chromophores have a very high absorbance around 680nm. Here is an example of an absorbance spectrum:
We followed the protocol that the Quail Lab used to extract the PCB from Spirulina, and were able to produce the following spectrum:
The concentration is found by this formula:
Engineering the PCB Synthesis Pathway into Yeast
The synthesis pathway of PCB begins with Heme to biliverdin (BV) until it finally becomes PCB. Many organisms already have a certain level of heme already. Our main goal was to create a strain of yeast that has the enzymes to be able to progress down the synthesis pathway. Below, you can see the synthesis pathway with the enzymes that we decided to become interested in.
HMX1 is a gene that we found that yeast already has. Enough BV is unfortunately not produced due to the fact that HMX1 is only transcribed under iron starvation conditions. For that reason we decided PCR HMX1 out of the genome and then place it on a plasmid with a much more powerful promoter, the promoter for ADH1. This plasmid is seen below.
The gene that we decided to follow was the PcyA gene, from the ___ genome. After looking at the codon usage charts for baker's yeast, and realized that we would need to do codon optimization to more effectively produce this enzyme in baker's yeast. The codon usage comparisons of PcyA before and after codon optimization, as done by GeneART, is seen below.
We synthesized this optimized PcyA using GeneART, and then cloned it into the plasmid, as shown below.
With these two plasmids, we were planning to transform these into a yeast strain with the proper plasmids to be able to complete the functional assay. Unfortunately, there were a lot of problems in creating this functional strain
