Team:Caltech/Project/Lactose intolerance
From 2008.igem.org
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RobertOvadia (Talk | contribs) |
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==Parts== | ==Parts== | ||
- | + | {{{!}} border="1" | |
- | + | ! Registry Number !! Plasmid !! Part !! Status | |
- | B0031 + LacY | + | {{!}}- |
- | B0033 + LacY | + | ! [http://partsregistry.org/Part:BBa_K137002 BBa_K137002] |
- | B0034-LacZ + B0015 | + | {{!}} pSB1A2 {{!}}{{!}} LacY {{!}}{{!}} Constructed |
- | B0031-LacY + B0015 | + | {{!}}- |
- | B0033-LacY + B0015 | + | ! [http://partsregistry.org/Part:BBa_K137126 BBa_K137126] |
- | J23113 + B0031-LacY-B0015 | + | {{!}} pSB1A2 {{!}}{{!}} Lysis Cassette {{!}}{{!}} Constructed |
- | + | {{!}}- | |
- | + | ! [http://partsregistry.org/Part:BBa_S03970 BBa_S03970] | |
- | J23113 + B0033-LacY-B0015 | + | {{!}} pSB1A2 {{!}}{{!}} B0031 + LacY {{!}}{{!}} Constructed |
- | J23106 + B0033-LacY-B0015 | + | {{!}}- |
- | + | ! [http://partsregistry.org/Part:BBa_S03971 BBa_S03971] | |
- | + | {{!}} pSB1A2 {{!}}{{!}} B0033 + LacY {{!}}{{!}} Constructed | |
- | + | {{!}}- | |
- | B0033-LacY | + | ! [http://partsregistry.org/Part:BBa_S03973 BBa_S03973] |
- | B0033-LacY | + | {{!}} pSB1AK3 {{!}}{{!}} B0034-LacZ + B0015 {{!}}{{!}} Constructed |
- | J23100 B0033-LacY SDM +2H | + | {{!}}- |
- | + | ! [http://partsregistry.org/Part:BBa_S04107 BBa_S04107] | |
- | B4 | + | {{!}} pSB1AK3 {{!}}{{!}} B0031-LacY + B0015 {{!}}{{!}} Constructed |
- | B0015 + A81 | + | {{!}}- |
- | B0015 + B4 | + | ! [http://partsregistry.org/Part:BBa_S04108 BBa_S04108] |
- | B0015 | + | {{!}} pSB1AK3 {{!}}{{!}} B0033-LacY + B0015 {{!}}{{!}} Constructed |
- | B0015 | + | {{!}}- |
- | + | ! [http://partsregistry.org/Part:BBa_S04109 BBa_S04109] | |
+ | {{!}} J61002 {{!}}{{!}} J23113 + B0031-LacY-B0015 {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04122 BBa_S04122] | ||
+ | {{!}} J61002 {{!}}{{!}} J23106 + B0031-LacY-B0015 {{!}}{{!}} Unavailable | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04123 BBa_S04123] | ||
+ | {{!}} J61002 {{!}}{{!}} J23100 + B0031-LacY-B0015 {{!}}{{!}} Unavailable | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04110 BBa_S04110] | ||
+ | {{!}} J61002 {{!}}{{!}} J23113 + B0033-LacY-B0015 {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04111 BBa_S04111] | ||
+ | {{!}} J61002 {{!}}{{!}} J23106 + B0033-LacY-B0015 {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04112 BBa_S04112] | ||
+ | {{!}} J61002 {{!}}{{!}} J23110 + B0033-LacY-B0015 {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04041 BBa_S04041] | ||
+ | {{!}} pSB1A2 {{!}}{{!}} B0033 + LacY +H {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04022 BBa_S04022] | ||
+ | {{!}} pSB1A2 {{!}}{{!}} B0033 + LacY +2H {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04113 BBa_S04113] | ||
+ | {{!}} pSB1AK3 {{!}}{{!}} B0033-LacY +H + B0034-LacZ-B0015 {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04054 BBa_S04054] | ||
+ | {{!}} pSB1AK3 {{!}}{{!}} B0033-LacY +2H + B0034-LacZ-B0015 {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04055 BBa_S04055] | ||
+ | {{!}} J61002 {{!}}{{!}} J23100 + B0033-LacY SDM +2H-B0034-LacZ-B0015 {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_K137124 BBa_K137124] | ||
+ | {{!}} pSB1A2 {{!}}{{!}} LacI Repressed Promoter A81 {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_K137125 BBa_K137125] | ||
+ | {{!}} pSB1A2 {{!}}{{!}} LacI Repressed Promoter B4 {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04114 BBa_S04114] | ||
+ | {{!}} pSB2K3 {{!}}{{!}} Lysis + B0015 {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04115 BBa_S04115] | ||
+ | {{!}} pSB2K3 {{!}}{{!}} B0015 + A81 {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04116 BBa_S04116] | ||
+ | {{!}} pSB2K3 {{!}}{{!}} B0015 + B4 {{!}}{{!}} Constructed | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04117 BBa_S04117] | ||
+ | {{!}} pSB2K3 {{!}}{{!}} B0015-A81 + Lysis-B0015 {{!}}{{!}} Constructed? | ||
+ | {{!}}- | ||
+ | ! [http://partsregistry.org/Part:BBa_S04118 BBa_S04118] | ||
+ | {{!}} pSB2K3 {{!}}{{!}} B0015-B4 + Lysis-B0015 {{!}}{{!}} Constructed? | ||
+ | {{!}}- | ||
+ | {{!}}} | ||
Revision as of 22:45, 22 October 2008
People
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Curing Lactose Intolerance
IntroductionThe gut flora of our digestive tract contains microorganisms that perform various useful functions for their hosts. Examples of such functions include growth inhibition of harmful microorganisms1, defense against the causes of many forms of Inflammatory Bowel Disease2, and the fermentation of carbohydrates and other molecules the human body cannot normally digest. Some bacterial strains, including the E. coli strain ‘Nissle 1917’, can persist in the gut of mice for months. Engineering these bacteria provide a new platform to treat various human diseases. System DesignLactose RegulatorThe first plasmid consists of a synthetic lactose operon under strong constitutive expression (Fig. 1). Our synthetic lactose operon encodes the ß-galactosidase LacZ and a lactose permease LacY. LacY is a membrane protein that actively transports lactose into the cell. Since most membrane proteins are toxic when overexpressed, we optimized our system to express appropriate levels of LacY without killing the cell. In the end, we want to express as much ß-galactosidase as we can to cleave as much lactose present in the large intestine. Lactose-Induced LysisThe second plasmid contains lactose inducible expression of λ phage lysis genes under a lactose inducible promoter (Fig. 2). To release ß-galactosidase, the cells will lyse when enough lactose is present in the cell to induce the expression of the lysis genes. It is important that the lactose-inducible promoter is tightly regulated since leaky expression will cause spontaneous lysis. To accomplish tight expression, specific lac promoters will keep our system from lysing5. In addition, the plasmid copy number remains low copy until induced with lactose, when the plasmid copy number increases to high copy. ResultsLactose RegulatorIn the first plasmid, it was discovered that LacY is toxic when overexpressed. To determine safe expression levels, constructs were built with varying levels of LacY expression. Six plasmids were built from combinations of three different promoters and two different ribosome binding sites (Fig. 3). The cells appeared to have died when expressing the strongest and medium strength promoters along with the strongest ribosome binding site. Our information was based off transformants, and the plates with no transformants were classified as dead cells due to high LacY expression. Based on these results, we decided to express our synthetic lac operon by combining the strongest constitutive promoter with the weakest RBS for LacY and the strongest RBS for LacZ, preventing LacY toxicity while expressing LacZ in large quantities. Lactose-Induced LysisOur second plasmid was not finished, but necessary parts have been cloned for future construction. There remains one more cloning step to place the lysis cassette into the vector containing the promoters. The single base mutations in the lysis cassette were not made, and if time allowed, they would have been completed as the final step. DiscussionOur final system was not completely finished; however, future construction will be minimal. The synthetic lac operon plasmid was completely constructed with the insertion of two histidyl residues in their appropriate locations. Our lactose-induced lysis plasmid was one cloning step away from being completed. The promoter and flanking terminator were cloned in parallel with the lysis cassette and a terminator. The last cloning step would be to ligate the two together. In addition, the two separate point mutations have not been made, and the final step in our cloning would be to add those mutations.
MethodsMethods can be found here Parts
References
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