Team:MIT/Projects/Project1

From 2009.igem.org

(Difference between revisions)
(→Engineering the PCB Synthesis Pathway into Yeast)
(→Engineering the PCB Synthesis Pathway into Yeast)
 
(25 intermediate revisions not shown)
Line 1: Line 1:
 +
[[Image:Bilibuddies_project1.png]]
__NOTOC__
__NOTOC__
-
<center>[[Team:MIT|Home]]|[[Team:MIT/Projects|Projects]]|[[Team:MIT/Protocols|Protocols]]|[[Team:MIT/PartsReg|Parts for Registry]]</center>
+
<center>[[Team:MIT|Home]] | [[Team:MIT/Projects|Projects]] | [[Team:MIT/Protocols|Protocols]] | [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=MIT Parts for Registry] | [[Team:MIT/References|References]]</center>
 +
<hr>
 +
 
 +
 
 +
 
 +
 
=Metabolic Engineering of PCB Synthesis in Yeast=
=Metabolic Engineering of PCB Synthesis in Yeast=
 +
[[Image:PCB_factory.jpg|center]]
 +
== Background ==
-
As shown in the image below, phycocyanobilin (PCB) plays a crucial part in the PhyB-PIF3 systemOnce 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.
+
Phycocyanobilin (PCB) is a chromophore essential for the physical interaction between two proteins, phytochrome PhyB and integrating factor PIF3.  PCB has two conformations: active and inactive.  In the inactive Pr conformation, PCB has the ability to absorb red light at a wavelength of 680 nm.  Once PCB absorbs red light, it switches to the active Pfr conformation and enables PhyB to bind to PIF3.  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 switches back to the inactive Pr conformation and causes dissociation of PhyB from PIF3. Shown below is the structure of PCB and how it switches between conformations.
-
<center>[[Image:Donor-recipient.jpg‎]]</center>
+
<center> [[Image:PCB_Structure.gif|350px]] [[Image:Pr_Pfr_Cartoon.png|170px]]</center>
-
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.
+
The synthesis pathway of PCB begins with Heme to biliverdin (BV) then to PCB. Heme is produced in a wide range of organisms. Our main goal is to create a strain of yeast able to progress down the PCB synthesis pathway as shown below.
-
<center>[[Image:PCB_Structure.gif|350px]] [[Image:Pr_Pfr_Cartoon.png|170px]]</center>
+
[[Image:PCB_Biosynthesis_Pathway.png|center|500px]]
-
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.
+
<center>''PCB biosynthesis pathway in Arabidopsis th. on the left and Synechocystis sp. on the right. Adapted from Gambetta, G. and Lagarias, JC., PNAS (2001)''</center>
 +
<br>
 +
In a 2002 ''Nature'' paper, the Quail Laboratory at University of California - Berekley used this PhyB-PIF3 system to induce ''LacZ'' expression.  The GAL4 DNA-binding domain was fused to PhyB while the GAL4 activation domain was fused to PIF3.  Once the system was pulsed with red light, ''LacZ'' expression was induced as shown below.
 +
[[Image:PhyB_PIF3_LacZ_Expression.png|center|450px]]
-
==Developing the Standard: PCB from Spirulina==
+
<center>''Adapted from Shimizu-Sato, S., et al., Nature Biotechnology (2002)''</center>
 +
<br><br>
-
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.
+
== Project Design ==
-
Chromophores have a very high absorbance around 680nmHere is an example of an absorbance spectrum:
+
As mentioned above, PCB plays a crucial part in the PhyB-PIF3 systemOur goal is to make yeast synthesize PCB, so that it will not need to be supplemented in the medium. In order to confirm the production of PCB by our engineered yeast strain, we needed a sensitive, functional assay to detect the presence of PCB.  We decided to use the two-hybrid system developed by the Quail lab, as illustrated below.  The protocol for this functional assay can be found [http://openwetware.org/wiki/Functional_Assay_for_PCB here].<br>
-
[[Image:Theoretical_PCB_Absorbance.png|center|250px]]
+
<center>[[Image:Donor-recipient.gif]]</center>
-
We followed the [http://openwetware.org/wiki/Extraction_of_PCB_from_Spirulina protocol that the Quail Lab] used to extract the PCB from ''Spirulina'', and were able to produce the following spectrum:
+
== Methods and Results ==
-
[[Image:Actual_PCB_Absorbance.png|center|250px]]
+
===Developing the Standard: PCB from Spirulina===
-
The concentration is found by this formula:
+
We also purified PCB from a natural source so that we would have a standard for the PCB synthesized in yeast.
 +
Phycocyanobilin (PCB) extracted from ''Spirulina'' is a commonly used standard, as ''Spirulina'' produces a large amount of chromophores.  We used ''Spirulina'' bought at Vitamin World, since ''Spirulina'' is commonly used as a dietary supplement.
-
[[Image:PCB_Conc_Formula.png|center]]
+
Typically, the chromophore has a very high absorbance around 680nm.  A model of what the absorbance spectrum should look like is on the left, while the actual absorbance spectrum of PCB we extracted from ''Spirulina'' is on the right. The extraction protocol can be found at [http://openwetware.org/wiki/Extraction_of_PCB_from_Spirulina protocol that the Quail Lab].
-
==Engineering the PCB Synthesis Pathway into Yeast==
+
<center>[[Image:Theoretical_PCB_Absorbance.png|250px]] [[Image:Actual_PCB_Absorbance.png|250px]] </center>
-
[[Image:PCB_factory.jpg|center]]
+
-
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.
 
-
[[Image:PCB_Biosynthesis_Pathway.png|center|500px]]
+
We were able to get a concentration of 11.214 mM in a 4.5 mL solution.
 +
 
 +
===Engineering the PCB Synthesis Pathway into Yeast===
-
<center>''Adapted from Gambetta, G. and Lagarias, JC., PNAS (2001)''</center>
 
-
''HMX1'' is a gene that we found that yeast already hasEnough 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 ''HMX1'' gene is already present in yeast.  However, not enough of biliverdin is typically produced in yeast because ''HMX1'' is only transcribed under iron starvation conditions.  Hence, we decided to clone ''HMX1'' from the genome and then overexpress it from a plasmid using the strong promoter from ''ADH1''.  This plasmid is diagramed below.
[[Image:HMX1_plasmid.png|center]]
[[Image:HMX1_plasmid.png|center]]
-
The gene that we decided to follow was the ''PcyA'' gene, from the ''Synechocystis sp.'' 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.
+
For the synthesis of PCB from biliverdin, we decided to clone the ''PcyA'' gene from the ''Synechocystis sp.'' cDNA library.  After looking at the codon usage charts for baker's yeast, we decided to codon optimize this gene to more effectively produce this enzyme in yeast.  The codon usage comparisons of ''PcyA'' before and after codon optimization (done by GeneART), is seen below.
[[Image:PcyA_Usage.png|center]]
[[Image:PcyA_Usage.png|center]]
-
We synthesized this optimized ''PcyA'' using GeneART, and then cloned it into the plasmid, as shown below.
+
We synthesized this optimized ''PcyA'' and cloned it into a vector, as shown below.
[[Image:PcyA_plasmid.png|center]]
[[Image:PcyA_plasmid.png|center]]
-
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, therefore we were unable to test whether or not these enzymes worked in yeast strain.  Yeast would produce PCB at such a lower level in comparison to ''Spirulina'' we can not isolate PCB using the same protocol.  Therefore the functional  assay is needed to see if yeast was being created or not.
+
Our next step is to transform both plasmids into a suitable yeast strain and employ the functional assay.  By comparison with a known concentration of purified PCB and commercially available biliverdin, we will be able to assess the effectiveness of these enzymes in yeast.

Latest revision as of 03:51, 22 October 2009

Bilibuddies project1.png

Home | Projects | Protocols | Parts for Registry | References



Metabolic Engineering of PCB Synthesis in Yeast

PCB factory.jpg

Background

Phycocyanobilin (PCB) is a chromophore essential for the physical interaction between two proteins, phytochrome PhyB and integrating factor PIF3. PCB has two conformations: active and inactive. In the inactive Pr conformation, PCB has the ability to absorb red light at a wavelength of 680 nm. Once PCB absorbs red light, it switches to the active Pfr conformation and enables PhyB to bind to PIF3. 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 switches back to the inactive Pr conformation and causes dissociation of PhyB from PIF3. Shown below is the structure of PCB and how it switches between conformations.

PCB Structure.gif Pr Pfr Cartoon.png

The synthesis pathway of PCB begins with Heme to biliverdin (BV) then to PCB. Heme is produced in a wide range of organisms. Our main goal is to create a strain of yeast able to progress down the PCB synthesis pathway as shown below.

PCB Biosynthesis Pathway.png
PCB biosynthesis pathway in Arabidopsis th. on the left and Synechocystis sp. on the right. Adapted from Gambetta, G. and Lagarias, JC., PNAS (2001)


In a 2002 Nature paper, the Quail Laboratory at University of California - Berekley used this PhyB-PIF3 system to induce LacZ expression. The GAL4 DNA-binding domain was fused to PhyB while the GAL4 activation domain was fused to PIF3. Once the system was pulsed with red light, LacZ expression was induced as shown below.

PhyB PIF3 LacZ Expression.png
Adapted from Shimizu-Sato, S., et al., Nature Biotechnology (2002)



Project Design

As mentioned above, PCB plays a crucial part in the PhyB-PIF3 system. Our goal is to make yeast synthesize PCB, so that it will not need to be supplemented in the medium. In order to confirm the production of PCB by our engineered yeast strain, we needed a sensitive, functional assay to detect the presence of PCB. We decided to use the two-hybrid system developed by the Quail lab, as illustrated below. The protocol for this functional assay can be found here.

Donor-recipient.gif

Methods and Results

Developing the Standard: PCB from Spirulina

We also purified PCB from a natural source so that we would have a standard for the PCB synthesized in yeast. Phycocyanobilin (PCB) extracted from Spirulina is a commonly used standard, as Spirulina produces a large amount of chromophores. We used Spirulina bought at Vitamin World, since Spirulina is commonly used as a dietary supplement.

Typically, the chromophore has a very high absorbance around 680nm. A model of what the absorbance spectrum should look like is on the left, while the actual absorbance spectrum of PCB we extracted from Spirulina is on the right. The extraction protocol can be found at protocol that the Quail Lab.

Theoretical PCB Absorbance.png Actual PCB Absorbance.png


We were able to get a concentration of 11.214 mM in a 4.5 mL solution.

Engineering the PCB Synthesis Pathway into Yeast

The HMX1 gene is already present in yeast. However, not enough of biliverdin is typically produced in yeast because HMX1 is only transcribed under iron starvation conditions. Hence, we decided to clone HMX1 from the genome and then overexpress it from a plasmid using the strong promoter from ADH1. This plasmid is diagramed below.

HMX1 plasmid.png

For the synthesis of PCB from biliverdin, we decided to clone the PcyA gene from the Synechocystis sp. cDNA library. After looking at the codon usage charts for baker's yeast, we decided to codon optimize this gene to more effectively produce this enzyme in yeast. The codon usage comparisons of PcyA before and after codon optimization (done by GeneART), is seen below.

PcyA Usage.png

We synthesized this optimized PcyA and cloned it into a vector, as shown below.

PcyA plasmid.png

Our next step is to transform both plasmids into a suitable yeast strain and employ the functional assay. By comparison with a known concentration of purified PCB and commercially available biliverdin, we will be able to assess the effectiveness of these enzymes in yeast.