Engineered Gut Microbiota
Vitamins
- Beta carotene
- Need others...
- Secretion? How are the vitamins readsorbed?
- Vitamin K - natural model
Lactose intolerance
- Secreted or intracellular?
- What's the proximate cause of the lactose intolerance phenotype?
Prophage targeting other bacteria
- lamB is sufficient for lambda infection.
- Looking at other receptors
- SPO2 is a lysogenic subtilis phage
- How do we get the phage inside the cell? Take a resistant strain (coli w/o lamB for lambda, coli for a subtilis phage), add the necessary receptor on a plasmid. Infect, select for lysogens. Grow under nonselective conditions, then counterselect for loss of the plasmid. Voila. Tetracycline can be counterselected[1].
Population Variation
- Slipped-strand mispairing (SSM)[2] can produce a heritable variation in the expression from a promoter. Roughly one in 1000 divisions produces a change in expression. Couple this expression to a selectable/counterselectable marker. Under any given condition (selection, say), the population thrives, but with a small group of the opposite phenotype (non-expressing). Switch conditions (to counterselecting), and the population can use these revertants to recover. The switching is stochastic by nature and can be directly compared to both natural [3] and synthetic [4] systems that utilize stochastic switching to adapt to variable and fluctuating environments.
- Also use FimE (below)?
Heavy Metal Chelation
ROS bursts
- H2O2 [5, 6]
- Turn on xanthine oxidase[7, 8] (or galactose oxidase[9]), turn off catalase
- Doug Tischer 03:23, 17 May 2008 (EDT)I like using xanthine oxidase because it can be suddenly "turned on" (really, changed from xanthine dehydrogenase to xanthine oxidase) through proteolytic cleavage. However, this is only true of mammalian xanthine oxidases. It is further complicated by the fact that xanthine oxidase can be revirsibly turned on/off by oxidation/reduction of some disulfide bonds. I'd be worried that because it is a mammalian protein with some disulfide bridges, that we wouldn't get good expression in bacteria. I tried to see if anyone has expressed it in E. coli, but haven't had any luck. There are bacterial versions of xanthine oxidase, but these can't be turned on/off like their mammalian cousins. We could try to make the bacterial xanthine oxidase turn on and off by preventing their natural dimerization by adding some interfering peptide sequence. This would be linked to the original protein by a linker domain that has a protease site. Once the protease is expressed, xanthine oxidase would be trimmed, it would dimerize, and we would get a sudden burst of H2O2. So this is where I'm stuck, since I don't have enough experience. Is it worth it to try and express the mammalian xanthine oxidase in E. coli and hope it can be done relatively easily or should I start looking into strategies for turning bacterial xanthine oxidase on and off?
- Trigger? [10, 11]
Conjugator of Death
- Engineered cell constitutively primed to conjugate with other bacteria. The transfered plasmid contains a collection of genes encoding for cytotoxic proteins (ie. ccdB, antimicrobial peptides, etc.)
- DB3.1 passing a plasmid with ccdB?
- Question of how well interspecies conjugation occurs and whether the natural gut microbiota would be susceptible
- Doug Tischer 03:06, 17 May 2008 (EDT)We could use the same trick the Peking team used to make a bacterial counter. The copy of the plasmid that our bacteria has would have transcriptional stops between the promoter and the toxic genes. Therefore no toxic products would be produced in our cells. However, upon conjugation the plasmid would only be partially replicated so as to omit the intervening transcriptional stops. Once the plasmid reaches the target cell the promoter would be in front of the toxic gene, producing the toxic proteins. Eventually, the target cell would die.
pH Control
References
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All Medline abstracts: PubMed | HubMed
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