Team:Guelph/Project

From 2008.igem.org

(Difference between revisions)
(→The Experiments)
(→The Experiments)
Line 63: Line 63:
=== The Experiments ===
=== The Experiments ===
-
1) beta carotene producing intestinal microbes and plant endophytes
+
1) Beta carotene producing intestinal microbes and plant endophytes
 +
 
 +
Tentative cloning strategy:
 +
 
 +
So what is the stategy we should follow? AThis is found in that
 +
Hydrogen synthetic operon paper.
 +
 
 +
http://www.ncbi.nlm.nih.gov/pubmed/17996187
 +
 
 +
Its specific for a bacterial synthetic
 +
operon and we don't have to rework things too hard in order to follow it.
 +
 
 +
I am making a Biobrick vector with a strong constitutive promoter from
 +
herbicide tolerant Amaranthus weeds which is unregulated (and therefore
 +
constitutive) in prokaryotes.  To do this we should PCR up the promoter
 +
from the pDSK-GFPuv plasmid, and put it into this:
 +
 
 +
http://partsregistry.org/wiki/index.php?title=Part:pSB1AK3
 +
 
 +
Likewise, I am PCRing up and inserting a restriction site optimized GFP
 +
into the plasmid. Then, we're ready to clone the carotenoid genes into
 +
the NdeI space in between promoter and GFP.
 +
 
 +
The gene order for the operon has already been optimized. This paper
 +
explains we should have E-B-I-Y. Check it out:
 +
 
 +
http://aem.asm.org/cgi/content/abstract/AEM.02268-06v1
 +
 
 +
So, the initial gene, crt-E on plasmid p3-10-10 will be amplified and
 +
appropriate flanking NdeI restriction sites introduced, alongside an
 +
internal ribosome binding site after the stop codon of the gene,
 +
followed by StuI (blunt RE) and AvrII. Cut the PCR product and the
 +
plasmid with NdeI, dephosphorylate the plasmid and ligate.
 +
 
 +
Now, to join Crt-B in there, cut it with SwaI (blunt RE) and AvrII,
 +
while cutting the plasmid with StuI and AvrII. Ligate. Again, there is a
 +
"AGGAGG" RBS directly after the stop codon which will promote
 +
translation of the following ORF. (note: CrtB has an internal StuI site
 +
which will have to be removed - there is a primer I included for this
 +
purpose, and would be used for PCR mediated mutagenesis and amplification
 +
followed by cutting with SmlI and StuI and ligating into the original PCR product)
 +
 
 +
Next comes Crt-I. This is done in the same way as above. cut it with
 +
SwaI and AvrII, while cutting the plasmid with StuI and AvrII. Ligate.
 +
(note: Crt-I has two PstI sites which really should be removed but don't
 +
have to if we don't have time - I have a technique I'd like to try to do
 +
this using some sort of nested PCR - there are a couple primers in the list
 +
which could be used to use to do this)
 +
 
 +
Finally, we insert Crt-Y. Cut it (PCR product) with SwaI and AvrII,
 +
while cutting the plasmid with StuI and AvrII. Ligate.
 +
 
 +
The GFP at the end should hopefully work as a reporter to let us know
 +
the transcript is OK, and the RBS sites are all lined up properly. If we
 +
want to add any more genes, we use the same protocol with SwaI, StuI and
 +
AvrII. Since these are all plasmid based PCRs, I expect it will be
 +
easier to get strong amplification than if we were using genomic DNA. In
 +
any case, I hope we could use proof reading enzymes for these PCRs - I
 +
have recently been turned on to Phusion from NEB. What do you use?
 +
 
2) RNAi inducing corn endophytes (BIGS)
2) RNAi inducing corn endophytes (BIGS)

Revision as of 06:10, 30 June 2008


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Contents

Overall project

Microbes are found in every nook and cranny of the entire planet, and multicellular organisms are no exception. Plants and animals are found to contain huge numbers of bacteria and fungi that help with nutrient absorption, produce beneficial compounds, fight off pathogens, or often are even pathogens themselves. We are interested in taking advantage of some of these microbes to engineer added functions into these microbes for the benefit or modification of the host organism. This might be called GM symbionts. On the human side, we would like to introduce the carotendoid metabolic genes from a well studied soil microbe called Erwinia urodevora into human intestinal microbes for production of the essential human nutrient, pro-vitamin A. Millions of humans suffer from vitamin A deficiencies across the world, resulting in blindness and death which could be mitigated by symbitic production of this important vitamin. A more basic project will focus on RNAi signal delivery by a corn plant endosymbiont to silence corn genes. Since microbes live in large stable populations within corn plants, it is believed that as individual bacteria grow and lyse within the plant host, they will release RNAi transcripts into the sensitive host during the entire life cycle of the plant, which will silence the targeted gene and show a phenotype indicating gene function. Bacterial Induced Gene Silencing (BIGS) will be a useful and quick alternative for plant functional genomic research.

Project Details

Part 2

The Experiments

1) Beta carotene producing intestinal microbes and plant endophytes

Tentative cloning strategy:

So what is the stategy we should follow? AThis is found in that Hydrogen synthetic operon paper.

http://www.ncbi.nlm.nih.gov/pubmed/17996187

Its specific for a bacterial synthetic operon and we don't have to rework things too hard in order to follow it.

I am making a Biobrick vector with a strong constitutive promoter from herbicide tolerant Amaranthus weeds which is unregulated (and therefore constitutive) in prokaryotes. To do this we should PCR up the promoter from the pDSK-GFPuv plasmid, and put it into this:

http://partsregistry.org/wiki/index.php?title=Part:pSB1AK3

Likewise, I am PCRing up and inserting a restriction site optimized GFP into the plasmid. Then, we're ready to clone the carotenoid genes into the NdeI space in between promoter and GFP.

The gene order for the operon has already been optimized. This paper explains we should have E-B-I-Y. Check it out: 

http://aem.asm.org/cgi/content/abstract/AEM.02268-06v1

So, the initial gene, crt-E on plasmid p3-10-10 will be amplified and appropriate flanking NdeI restriction sites introduced, alongside an internal ribosome binding site after the stop codon of the gene, followed by StuI (blunt RE) and AvrII. Cut the PCR product and the plasmid with NdeI, dephosphorylate the plasmid and ligate.

Now, to join Crt-B in there, cut it with SwaI (blunt RE) and AvrII, while cutting the plasmid with StuI and AvrII. Ligate. Again, there is a "AGGAGG" RBS directly after the stop codon which will promote translation of the following ORF. (note: CrtB has an internal StuI site which will have to be removed - there is a primer I included for this purpose, and would be used for PCR mediated mutagenesis and amplification followed by cutting with SmlI and StuI and ligating into the original PCR product)

Next comes Crt-I. This is done in the same way as above. cut it with SwaI and AvrII, while cutting the plasmid with StuI and AvrII. Ligate. (note: Crt-I has two PstI sites which really should be removed but don't have to if we don't have time - I have a technique I'd like to try to do this using some sort of nested PCR - there are a couple primers in the list which could be used to use to do this)

Finally, we insert Crt-Y. Cut it (PCR product) with SwaI and AvrII, while cutting the plasmid with StuI and AvrII. Ligate.

The GFP at the end should hopefully work as a reporter to let us know the transcript is OK, and the RBS sites are all lined up properly. If we want to add any more genes, we use the same protocol with SwaI, StuI and AvrII. Since these are all plasmid based PCRs, I expect it will be easier to get strong amplification than if we were using genomic DNA. In any case, I hope we could use proof reading enzymes for these PCRs - I have recently been turned on to Phusion from NEB. What do you use?


2) RNAi inducing corn endophytes (BIGS)

Part 3

Results

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