IGEM:Caltech/2008/Project: Difference between revisions
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==Engineered Gut Microbiota== | ==Engineered Gut Microbiota== | ||
*[[/Supplies|Strains we're requesting]] | |||
*[[/Tasks|Stuff to do]] | |||
[[User:Josh K. Michener|Josh K. Michener]] 20:49, 4 June 2008 (UTC): Okay, my grand (partly) unified (and only partly feasible) vision: We have the cells metabolizing lactose and feeding that carbon into central metabolism. The excess flux is diverted into folate biosynthesis (rather than lactic/acetic acid production). The folate is periodically liberated from cells by inducing prophage expression - the prophage lyses the cell and as a side effect releases the vitamins. I still can't work Doug's project into it, though. | |||
*'''[[User:Cbeisel|Cbeisel]] 04:33, 16 June 2008 (UTC)''':How do we envision our engineered gut bacteria will be used? One idea is distributing it in yogurt and baby formula. Thinking about it from a marketing standpoint may help develop the final story. | |||
===[[/Vitamins|Vitamins]]=== | ===[[/Vitamins|Vitamins]]=== | ||
*Vitamin choice: [http://books.nap.edu/openbook.php?isbn=0309065542 General Reference] | *Vitamin choice: [http://books.nap.edu/openbook.php?isbn=0309065542 General Reference] | ||
**Folate: 400-600ug RDA. Naturally produced by E. coli. Six enzymatic steps. Decent bioavailability (not enough for your whole RDA, but a measurable contribution). I haven't found any indication of the rate limiting step.<cite>asrar, camilo, bermingham, gabelli, sybesma2, zhu</cite>. Folate deficiency in pregnant women can cause birth defects. | **Folate: 400-600ug RDA. Naturally produced by E. coli. Six enzymatic steps. Decent bioavailability (not enough for your whole RDA, but a measurable contribution). I haven't found any indication of the rate limiting step.<cite>asrar, camilo, bermingham, gabelli, sybesma2, zhu</cite>. Folate deficiency in pregnant women can cause birth defects. | ||
***[[User:Victoria Hsiao|Victoria Hsiao]] 10:13, 11 June 2008 (UTC)In Zhu et al <cite>zhu</cite>, they cited a paper that suggested folK was the rate limiting enzyme (pyrophosphokinase). | |||
***[[User:Victoria Hsiao|Victoria Hsiao]] 10:13, 11 June 2008 (UTC)What are the differences between the pathway in e coli and that in L.lactus or B.subtilis? I'm having a hard time finding any papers that have the folate synthesis pathway in e coli.. I found this site that has the pathway mapped out, [http://www.kegg.jp/dbget-bin/get_pathway?org_name=eco&mapno=00790], but would the order that the enzymes appear in correspond to the order that their corresponding genes need to be in in the gene cluster? | |||
***[[User:Victoria Hsiao|Victoria Hsiao]] 10:13, 11 June 2008 (UTC) In Sybesma et al <cite>sybesma1</cite> they suggest that overexpression of folKE and folC is best for the intracellular accumulation of folate in L.lactis. folKE(2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase & GTP cyclohydrolase I), folC (polyglutamylfolate synthetase). They also noted that overexpression of GTP cyclohydrase I can be effective for increasing flux since it is the first enzyme in the pathway. | |||
****[[User:Josh K. Michener|Josh K. Michener]] 19:52, 11 June 2008 (UTC): Look at Bermingham et al. <cite>bermingham</cite> Figure 1 for the E. coli folate biosynthesis pathway. Keep in mind that it introduces PABA and glutamate without explicitly listing their synthetic pathways. I think the subtilis folK is the coli HPPK. If I read it right, mtrE is the same as folE, which in coli is GTPCH1. | |||
***[[User:Victoria Hsiao|Victoria Hsiao]] 09:59, 15 June 2008 (UTC) Wegkamp et al used the folate gene cluster from L.lactis to turn a folate consuming bacteria into a folate producing one! <cite>wegkamp1</cite> | |||
**** [[User:Victoria Hsiao|Victoria Hsiao]] 09:59, 15 June 2008 (UTC) I've found the corresponding genes for E. coli, but maybe we should also use the L.lactis gene cluster since it would be less regulated? | |||
***[[User:Victoria Hsiao|Victoria Hsiao]] 09:59, 15 June 2008 (UTC) The same group also characterized the pabA,pabB, and pabC genes from L.lactis and determined and that are 3 are important and necessary in the production of folate. <cite>wegkamp2</cite> | |||
**[http://www.ncbi.nlm.nih.gov/pubmed/11676567?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Beta carotene]: Need 3-6mg per day[http://ods.od.nih.gov/factsheets/vitamina.asp]. Unclear how much the Keasling lab made, since it's all reported as relative production<cite>smolke2001</cite>. | **[http://www.ncbi.nlm.nih.gov/pubmed/11676567?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Beta carotene]: Need 3-6mg per day[http://ods.od.nih.gov/factsheets/vitamina.asp]. Unclear how much the Keasling lab made, since it's all reported as relative production<cite>smolke2001</cite>. | ||
***'''[[User:Cbeisel|Cbeisel]] 13:08, 2 June 2008 (EDT)''':We currently have some of the plasmids used in the paper, so it shouldn't be difficult to initially produce Beta carotene and begin optimizing production. | ***'''[[User:Cbeisel|Cbeisel]] 13:08, 2 June 2008 (EDT)''':We currently have some of the plasmids used in the paper, so it shouldn't be difficult to initially produce Beta carotene and begin optimizing production. | ||
***[[User:Victoria Hsiao|Victoria Hsiao]] 17:08, 4 June 2008 (UTC)Just to make sure I understood the paper correctly, they were able to vary the production rate of the beta-carotene by changing the length of the hairpin 5' of the crtI? | ***[[User:Victoria Hsiao|Victoria Hsiao]] 17:08, 4 June 2008 (UTC)Just to make sure I understood the paper correctly, they were able to vary the production rate of the beta-carotene by changing the length of the hairpin 5' of the crtI? | ||
***[[User:Josh K. Michener|Josh K. Michener]] 21:58, 4 June 2008 (UTC): They were changing the relative stability of the mRNAs, which changes the relative expression of the genes in the operon. | |||
**Biotin: 30 ug RDA. The Liao lab got 100ug/L in an engineered pathway<cite>bernstein</cite> | **Biotin: 30 ug RDA. The Liao lab got 100ug/L in an engineered pathway<cite>bernstein</cite> | ||
***[[User:Victoria Hsiao|Victoria Hsiao]] 21:47, 3 June 2008 (CDT)From Wikipedia: "Deficiency is extremely rare as intestinal bacteria generally produce an excess of the body's daily requirement. For that reason statutory agencies in many countries (e.g. the Australian Department of Health and Aging) do not prescribe a recommended daily intake." [http://en.wikipedia.org/wiki/Biotin] Would that make biotin production not as relevent a concern? | ***[[User:Victoria Hsiao|Victoria Hsiao]] 21:47, 3 June 2008 (CDT)From Wikipedia: "Deficiency is extremely rare as intestinal bacteria generally produce an excess of the body's daily requirement. For that reason statutory agencies in many countries (e.g. the Australian Department of Health and Aging) do not prescribe a recommended daily intake." [http://en.wikipedia.org/wiki/Biotin] Would that make biotin production not as relevent a concern? | ||
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*****[[User:Victoria Hsiao|Victoria Hsiao]] 16:36, 4 June 2008 (UTC)Could we just make it a timing thing, and not have a switch at all? i.e the lysis gene and the vitamin production gene are both constantly being expressed, but at a rate such that the cell does not actually lyse until a time when we're pretty sure the bacteria has already produced a significant amount of vitamin. So then we'd just have to figure out the rate of vitamin production, and then engineer the rate of lysis gene expression to match that time. Could we use the hairpin approach used in this paper <cite>smolke2001</cite> to match the relative production rates? | *****[[User:Victoria Hsiao|Victoria Hsiao]] 16:36, 4 June 2008 (UTC)Could we just make it a timing thing, and not have a switch at all? i.e the lysis gene and the vitamin production gene are both constantly being expressed, but at a rate such that the cell does not actually lyse until a time when we're pretty sure the bacteria has already produced a significant amount of vitamin. So then we'd just have to figure out the rate of vitamin production, and then engineer the rate of lysis gene expression to match that time. Could we use the hairpin approach used in this paper <cite>smolke2001</cite> to match the relative production rates? | ||
****[[User:Josh K. Michener|Josh K. Michener]] 15:26, 2 June 2008 (EDT): Looks like there's a biotin sensor<cite>cronan</cite>. Still looking for a folate one. | ****[[User:Josh K. Michener|Josh K. Michener]] 15:26, 2 June 2008 (EDT): Looks like there's a biotin sensor<cite>cronan</cite>. Still looking for a folate one. | ||
*[[User:Josh K. Michener|Josh K. Michener]] 17:17, 9 June 2008 (UTC): As a fall back plan - do we necessarily need to sense when to trigger lysis? At steady state, the vitamin concentration is only going to vary by a factor of two (just after division to just before division). So we could randomly trigger lysis and get pretty good release. | |||
**[[User:Victoria Hsiao|Victoria Hsiao]] 10:47, 11 June 2008 (UTC) In this paper, Templin et al [http://www.nature.com/emboj/journal/v18/n15/pdf/7591826a.pdf] deleted the gene ldcA which encodes a cytoplasmic L,D-carboxypeptidase. The mutant with the ldcA deletion was unable to make the pentapeptide precursor needed to recycle the murein(peptidoglycan) in the cell wall) and instead steadily accumulated tetrapeptide in the cytoplasm. As a result of the weakened cell wall, this lead to spontaneous autolysis during the stationary growth phase. This sounds promising, as long as most of the folate production occurred prior to reaching the stationary growth phase. Although i don't know how we'd ensure that enough bacteria lived on to maintain a stable population. Also, ampD is the gene that controls the murein recycling, so maybe we could also do something that that; when they deleted both ldcA and ampD, the e coli did not produce the mutant tetrapeptide. | |||
***[[User:Josh K. Michener|Josh K. Michener]] 20:01, 11 June 2008 (UTC): Question - do we think that the cells would reach stationary phage in the gut? Or, more correctly, would they have sufficient stress to cause autolysis? | |||
***[[User:Josh K. Michener|Josh K. Michener]] 20:01, 11 June 2008 (UTC): It looks like we have several options for lysis - prophage, the T4 holin, the T7 lysozyme, the ldcA deletion. I'd probably try several options. | |||
****[[User:Josh K. Michener|Josh K. Michener]] 23:02, 12 June 2008 (UTC): This paper [http://dx.doi.org/10.1016/S0922-338X(97)80357-1] looks at expressing the holin by itself, and sees nearly complete lysis within 30 minutes. | |||
*** *'''[[User:Cbeisel|Cbeisel]] 00:28, 13 June 2008 (UTC)''':Here's a paper that shows 30-60 min lysis when various holin variants are expressed from a plasmid <cite>ramanculov2001</cite>[http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T39-42JHDKP-3-F&_cdi=4941&_user=1010281&_orig=search&_coverDate=03%2F07%2F2001&_sk=997349998&view=c&wchp=dGLbVtb-zSkWz&md5=e3bb3fbc2dddeccefb09c1c0fa0a23d9&ie=/sdarticle.pdf]. At first glance, it appears we can make point mutations or truncations that increase the lysis time...assuming that's of any use to us. | |||
===Lactose intolerance=== | ===[[/Lactose intolerance|Lactose intolerance]]=== | ||
*Secreted or intracellular?<cite>jiang, devrese</cite> | *Secreted or intracellular?<cite>jiang, devrese</cite> | ||
**'''[[User:Robert Ovadia|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. | **'''[[User:Robert Ovadia|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. | ||
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***'''[[User:Robert Ovadia|Robert]] 17:27, 3 June 2008 (CDT)''':I was reading on p. 946 in the section Regulation of Carbon Metabolism in Gram-Negative Enteric Bacteria, and they share an idea that might help our strain. They showed ptsHI mutants, more specifically, mutations in the crr gene, which encodes for EIIA(GLUC) allowed the strain to grow on non-PTS carbon sources, ours being lactose. It also made it not able to grow in the PTS carbon sources, such as glucose. Later, they discussed the phosphorylation and dephosphorylation of EIIA(GLUC) and P~EIIA(GLUC). Unphosphorylated EIIA(GLUC) can bind and inhibit our lacY protein, along with other non-PTS carbon sources. Is it possible to block the binding of EIIA(GLUC), or keep EIIA(GLUC) ALWAYS phosphorylated? | ***'''[[User:Robert Ovadia|Robert]] 17:27, 3 June 2008 (CDT)''':I was reading on p. 946 in the section Regulation of Carbon Metabolism in Gram-Negative Enteric Bacteria, and they share an idea that might help our strain. They showed ptsHI mutants, more specifically, mutations in the crr gene, which encodes for EIIA(GLUC) allowed the strain to grow on non-PTS carbon sources, ours being lactose. It also made it not able to grow in the PTS carbon sources, such as glucose. Later, they discussed the phosphorylation and dephosphorylation of EIIA(GLUC) and P~EIIA(GLUC). Unphosphorylated EIIA(GLUC) can bind and inhibit our lacY protein, along with other non-PTS carbon sources. Is it possible to block the binding of EIIA(GLUC), or keep EIIA(GLUC) ALWAYS phosphorylated? | ||
*** *'''[[User:Cbeisel|Cbeisel]] 18:24, 3 June 2008 (CDT)''':You have the right idea. EIIA(GLUC) will bind lacY, thereby inhibiting transport when glucose is present. Mutating or deleting EIIA would allow uninhibited transport of lactose, although the cells can't uptake glucose. Another option is mutating lacY to prevent the inhibitory binding interaction. It appears a few groups have identified point mutations in lacY that abolish EIIA inhibition without affecting lactose transport. This paper is a good start <cite>Hoischen1996</cite> and may be a simple solution to this aspect of lactose intolerance. | *** *'''[[User:Cbeisel|Cbeisel]] 18:24, 3 June 2008 (CDT)''':You have the right idea. EIIA(GLUC) will bind lacY, thereby inhibiting transport when glucose is present. Mutating or deleting EIIA would allow uninhibited transport of lactose, although the cells can't uptake glucose. Another option is mutating lacY to prevent the inhibitory binding interaction. It appears a few groups have identified point mutations in lacY that abolish EIIA inhibition without affecting lactose transport. This paper is a good start <cite>Hoischen1996</cite> and may be a simple solution to this aspect of lactose intolerance. | ||
**'''[[User:Robert Ovadia|Robert]] 18:42, 4 June 2008 (UTC)''': I was looking for enzymes, to convert H2 or methane, with the sites Doug shared w/ the team, and the only enzyme I found was the one he pointed out in his e-mail, methane hydroxylase. I am wondering if this approach using enzymes is possible? Also, for the reaction with methane hydroxylase, methanol is produced. Would methanol cause any other problems? I tried google and pubmed, but I wound up empty. | |||
***[[User:Josh K. Michener|Josh K. Michener]] 20:45, 4 June 2008 (UTC): If I understand correctly, you're proposing to use a methane monooxygenase (MMO), correct? That's basically impossible to express in E. coli - it's a multi-component system from an archaea. We might be able to use the hydrogen, but methane is probably impossible. | |||
****'''[[User:Robert Ovadia|Robert]] 20:49, 4 June 2008 (UTC)''': Oh ok. I really wasn't sure about how we would use enzymes that convert methane and H2 as one of the possible ways to target lactose intolerance. Should I disregard that goal? | |||
*****[[User:Josh K. Michener|Josh K. Michener]] 20:50, 4 June 2008 (UTC): I'd see if you can find a way to use the hydrogen. | |||
***'''[[User:Robert Ovadia|Robert]] 00:56, 15 June 2008 (UTC)''':For glucose uptake in the large intestine (in a canine), one paper showed that when adrenaline is present, there is a 150% increase in glucose uptake. The main idea is glucose uptake takes place in the large intestine. The numbers they found were 28.28 ± 20mg/min without adrenaline present.[http://www.bioline.org.br/request?md01064 Glucose uptake in dogs] | |||
****[[User:Josh K. Michener|Josh K. Michener]] 01:05, 15 June 2008 (UTC): From some poking around, it looks like milk is about 5% lactose (50mg/mL)[http://jds.fass.org/cgi/reprint/83/9/1939.pdf]. So an 8oz glass of milk has ~12g of lactose. At the dog's rate (keep in mind humans would be faster), that would take 6.7 hours to absorb. Can we find a ratio of the surface area of a dog's large intestine to a human? | |||
***'''[[User:Robert Ovadia|Robert]] 01:55, 15 June 2008 (UTC)''':Here is an approximate of the human intestine. It's about 1.5m long, and a diameter of about 2.5 inches. After calculation, SA is appx. 3000 sq cm. Although in this paper, [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9308197&dopt=Abstract Rat Microvilli] showing that the microvilli of a rat's intestine enlarges the SA by as large as a factor of 20, so 6 sq meters is a rough estimate for a human. For dogs, an appx length of a regular sized 40 lb dog is 16 inches. If we assume both the dog's and human's large colon are proportional, it's about 1/4 the size. | |||
****[[User:Josh K. Michener|Josh K. Michener]] 04:09, 15 June 2008 (UTC): Okay, that gives us ~100 minutes to absorb the lactose from a glass of milk. So our timescales are similar (though it would be nice if absorption were a little faster). | |||
***'''[[User:Robert Ovadia|Robert]] 02:10, 15 June 2008 (UTC)''': One question I will try to research is how much lactose would cause producing methane gas, and stomach pain. A few lactose intolant people I know are not able to consume milk, but they are able to consume yogurt with no problem. If we can reduce the amount of lactose being metabolized and converted into H2 and Methane, to an amount that doesn't produce enough methane to have an effect, would this be reasonable? | |||
*** *'''[[User:Cbeisel|Cbeisel]] 22:32, 15 June 2008 (UTC)''':Keep in mind that we intend to release B-gal into the gut to quickly break down lactose into glucose and galactose. Can you find anything on what an excess of glucose in the large intestine does? As long as B-gal breaks lactose down fast enough, gut adsorption may be significant enough to prevent major side effects. As a side note, feeding our gut microbes can be a good thing [http://en.wikipedia.org/wiki/Prebiotic_(nutrition)] as long as it happens on a somewhat regular basis. | |||
****'''[[User:Robert Ovadia|Robert]] 05:00, 16 June 2008 (UTC)''':I was not able to find anything about excess glucose in the large intestine, but I found a lot of pages about diabetes. I'm going to look at what causes diabetes and see if the excess glucose will have any effect (I don't know much about diabetes but I'll present what I find at our meeting tomorrow). | |||
*Adaptation <cite>hertzler</cite> | |||
===Prophage targeting other bacteria=== | ===Prophage targeting other bacteria=== | ||
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*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<cite>bochner</cite>. | *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<cite>bochner</cite>. | ||
*Works in cows<cite>sheng</cite> | *Works in cows<cite>sheng</cite> | ||
*We need to select a suitable phage for the project. For convenience, here is a list of phages we can order from atcc: [http://www.atcc.org/ATCCAdvancedCatalogSearch/tabid/112/Default.aspx] | |||
*SPO1, Phie, and SP8 all belong to the same family [http://www.ncbi.nlm.nih.gov/pubmed/1444272?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]. | |||
They have linear, double stranded DNA however, it appears that they have thymine replaced with hydroxymethyluracil. It is not well characterized how well this works with cloning... | |||
*SPP1 looks promising even though it is not a propahge. It possesses double stranded linear DNA. Furthermore, this paper [http://www.springerlink.com/content/m3x89ug4182g6rr7/] describes creating a fusion virus of SPP1/lamda. They can infect E. Coli, and express both lamda and SPP1 genes within E. Coli. This shows some promise and offers new ideas. Perhaps we can construct a fusion phage? | |||
*SPP1 complete genome can be found here:[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=111146921] and restriction mapping can be found here: [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=525811]. | |||
*We need to transform large plasmids, transformation efficiency generally goes down, however, electroporation conditions can be used to compensate. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=306974] | |||
===Population Variation=== | ===Population Variation=== | ||
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#cronan pmid=2667763 | #cronan pmid=2667763 | ||
#Hoischen1996 pmid=8830713 | #Hoischen1996 pmid=8830713 | ||
#hertzler pmid=8694025 | |||
#ramanculov2001 pmid=11255004 | |||
#raden pmid=6090713 | |||
#gemski pmid=4564201 | |||
#devries pmid=6091132 | |||
#bielke pmid=18029799 | |||
#deng pmid=8144476 | |||
#baxa pmid=8889178 | |||
#elledge pmid=2985547 | |||
#lindsey pmid=1534329 | |||
#vanbelkum pmid=9618442 | |||
#wegkamp1 pmid=15128580 | |||
#wegkamp2 pmid=17308179 | |||
</biblio> | </biblio> | ||
|} | |} |
Latest revision as of 10:53, 24 June 2008
Engineered Gut MicrobiotaJosh K. Michener 20:49, 4 June 2008 (UTC): Okay, my grand (partly) unified (and only partly feasible) vision: We have the cells metabolizing lactose and feeding that carbon into central metabolism. The excess flux is diverted into folate biosynthesis (rather than lactic/acetic acid production). The folate is periodically liberated from cells by inducing prophage expression - the prophage lyses the cell and as a side effect releases the vitamins. I still can't work Doug's project into it, though.
Vitamins
Lactose intolerance
Prophage targeting other bacteria
They have linear, double stranded DNA however, it appears that they have thymine replaced with hydroxymethyluracil. It is not well characterized how well this works with cloning...
Population Variation
ROS bursts
References
|