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Team:Guelph/Project
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The crt-B PCR product is flanked with XbaI and AvrII, with an internal SpeI site after the stop codon of the crt-B gene. To insert the crt-B after crt-E, we will cut it with XbaI and AvrII, while cutting the plasmid with SpeI and AvrII. The complementary pairing between SpeI and XbaI's sticky ends will again allow them to ligate, destroying the Spe and Xba sites in the process. | The crt-B PCR product is flanked with XbaI and AvrII, with an internal SpeI site after the stop codon of the crt-B gene. To insert the crt-B after crt-E, we will cut it with XbaI and AvrII, while cutting the plasmid with SpeI and AvrII. The complementary pairing between SpeI and XbaI's sticky ends will again allow them to ligate, destroying the Spe and Xba sites in the process. | ||
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Next comes Crt-I. This is done in the same way as above, as it is also flanked with XbaI and AvrII with an internal SpeI site. Once again, the PCR product is cut with XbaI and AvrII, while the plasmid is cut with SpeI and AvrII. Ligate. | Next comes Crt-I. This is done in the same way as above, as it is also flanked with XbaI and AvrII with an internal SpeI site. Once again, the PCR product is cut with XbaI and AvrII, while the plasmid is cut with SpeI and AvrII. Ligate. | ||
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Finally, we insert Crt-Y. Cut it (PCR product) with XbaI and AvrII, while cutting the plasmid with SpeI and AvrII. Ligate. | Finally, we insert Crt-Y. Cut it (PCR product) with XbaI and AvrII, while cutting the plasmid with SpeI 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 | + | 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 XbaI, SpeI 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. |
| + | |||
| + | Assay for beta-carotene production. | ||
| + | |||
| + | Transfer to gram positive and gram negative plasmids. | ||
| + | |||
| + | Electroporate into different probiotic strains. | ||
| + | |||
| + | Monitor beta carotene production and GFP in biological system/model. | ||
| + | |||
| + | Celebrate a succesful new technology for improved human health! | ||
| + | |||
| + | |||
2) RNAi inducing corn endophytes (BIGS) | 2) RNAi inducing corn endophytes (BIGS) | ||
| + | |||
| + | In our heads if not on the paper yet. | ||
== Results == | == Results == | ||
Revision as of 19:08, 8 July 2008
Contents |
Project Abstract
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.
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(Or you can choose different headings. But you must have a team page, a project page, and a notebook page.)
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
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 (introducing an SpeI site after the native NdeI), 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
The initial gene, crt-E, on plasmid p3-10-10 will be amplified and appropriate flanking NdeI and XbaI restriction sites introduced, as well as a SpeI and an AvrII site after the stop codon of the gene. There is also a ribosome binding site present in front of each of the carotenoid genes, and thus we will conserve them in our cloning strategy. We will cut the plasmid with NdeI and SpeI, and the PCR product with NdeI and XbaI. We will then dephosphorylate the plasmid and ligate. SpeI and Xba I being sticky-end cutters with complementary overhangs, their respective cut ends will ligate to each other. This will destroy the SpeI site on the plasmid and the XbaI site on the PCR product, but these complementary sticky ends have the benefit of decreasing the rate of background ligations; the complementary sticky ends act as 'heat seeking missiles', seeking out their complementary mates and ignoring non-complementary overhangs or blunt end.
The crt-B PCR product is flanked with XbaI and AvrII, with an internal SpeI site after the stop codon of the crt-B gene. To insert the crt-B after crt-E, we will cut it with XbaI and AvrII, while cutting the plasmid with SpeI and AvrII. The complementary pairing between SpeI and XbaI's sticky ends will again allow them to ligate, destroying the Spe and Xba sites in the process.
Next comes Crt-I. This is done in the same way as above, as it is also flanked with XbaI and AvrII with an internal SpeI site. Once again, the PCR product is cut with XbaI and AvrII, while the plasmid is cut with SpeI 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 XbaI and AvrII, while cutting the plasmid with SpeI 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 XbaI, SpeI 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.
Assay for beta-carotene production.
Transfer to gram positive and gram negative plasmids.
Electroporate into different probiotic strains.
Monitor beta carotene production and GFP in biological system/model.
Celebrate a succesful new technology for improved human health!
2) RNAi inducing corn endophytes (BIGS)
In our heads if not on the paper yet.
