IGEM:Caltech/2008/Project

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iGEM 2008

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Engineered Gut Microbiota Vitamins Beta carotene Need others... Biotin[1] Vitamin D Vitamin E Folate[2, 3, 4, 5] Secretion? How are the vitamins readsorbed? Vitamin K - natural model[6] Lactose intolerance Secreted or intracellular?[7, 8] Robert 22:18, 22 May 2008 (EDT):For article 7, does B. longum contain the beta-gal gene? Or are they testing another way to reduce lactose by using B. longum? I googled the strain and I couldn't find much about it, but that it does ferment sugars into lactic acid, so I am guessing it has the gene, or one of similar kind. What's the proximate cause of the lactose intolerance phenotype? Usually, lactose is split into glucose and galactose by lactase present in the villi of the small intestine. If lactase is not present, the lactose continues into the colon, where it is metabolized by gut flora, resulting in in vivo fermentation. Wikipedia Josh K. Michener 12:25, 19 May 2008 (EDT): Lactose causes two problems: first, metabolism into H2 (and possibly further into methane) or it changes the osmolarity of the gut and causes diarrhea [1]. So there are a number of directions we can attack this from - consume H2, do a better job of making lactase, digesting the glucose and galactose first, importing and sequestering the lactose, etc. The natural pathway seems to be sugar->lactate/acetate->H2->methane. So if we can get a good aerobic culture going, that might be enough. Our lactase-producing bacteria would have to somehow outcompete the lactose-metabolizing bacteria in the colon. It would at least have to get to the lactose first, so that the other bacteria don't get a chance to ferment it. Or it could produce so much extracellular lactase that any lactose entering the colon would immediately be broken down. Would we want our bacteria to live in the small intestine? 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[9]. Population Variation Slipped-strand mispairing (SSM)[10] 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 [11] and synthetic [12] systems that utilize stochastic switching to adapt to variable and fluctuating environments. Also use FimE (below)? Heavy Metal Chelation Need details here ROS bursts ROS H2O2 [13, 14] Turn on xanthine oxidase[15, 16] (or galactose oxidase[17]), 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? References? Yamaguchi et al[15] showed that they could express the human XO in E. coli, but they only looked for activity in vitro. Not clear that they'd have activity in vivo. Doug Tischer 05:46, 18 May 2008 (EDT)Looks like it is very tricky to express mammalian xanthine oxidase in bacteria, mostly because of the molybdenum center. The paper above reported that only 4% of their expressed xanthine oxidase was active. I'm now thinking it might be best to take an enzyme that produces H2O2 that is naturally found in bacteria and is a homodimer (like glucose oxidase, I think). Then, express one of the monomers as a fusion protein on the cytoplasmic C-terminal tail of a integral membrane protein, linked by a linker region with a protease site. This setup would prevent dimerization until the monomers are released into the cytoplasm when the protease is expressed Josh K. Michener 14:59, 18 May 2008 (EDT): Remember, try to make this an incremental progression, not a moon shot. Also, keep in mind that the simplest solution is the most likely to work. So the first step would be expression of an oxidase and measurable production of peroxide in vivo. I'd probably take a couple different oxidases (try the galactose oxidase[17], for instance, in addition to a glucose oxidase - that one's upstairs in the Arnold Lab) and just see how much peroxide you can make. Then see how well you can regulate it just with transcriptional regulation - try it in BL21(DE3)pLysS, for instance. You could probably do that in parallel with the protein engineering for your protease cascade, but don't rely on any given thing working. Trigger by conjugation? [18, 19] 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. 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