Team:Caltech

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==Engineering multi-functional probiotic bacteria==
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[[Image:Gut_flora_color.png|right|thumb|200px|Engineered gut flora]]
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The human gut houses a diverse collection of microorganisms, with important implications for the health and welfare of the host. We aim to engineer a member of this microbial community to provide innovative medical treatments. Our work focuses on four main areas: (1) pathogen defense either by expression of [[Team:Caltech/Project/Phage_Pathogen_Defense|<font style="color:#BB4400">pathogen-specific bacteriophage</font>]] or by targeted bursts of [[Team:Caltech/Project/Oxidative_Burst|<font style="color:#BB4400">reactive oxygen species</font>]]; (2) prevention of birth defects by [[Team:Caltech/Project/Vitamins|<font style="color:#BB4400">folate over-expression</font>]] and delivery; (3) treatment of [[Team:Caltech/Project/Lactose_intolerance|<font style="color:#BB4400">lactose intolerance</font>]] by cleaving lactose to allow absorption in the large intestine; and (4) [[Team:Caltech/Project/Population_Variation|<font style="color:#BB4400">regulation</font>]] of these three treatment functions to produce renewable subpopulations specialized for each function. Our research demonstrates that synthetic biology techniques can be used to modify naturally occurring microbial communities for applications in biomedicine and biotechnology.
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We aim to engineer a probiotic bacterium to improve its medical applications. Our work focuses on four main areas: (1) pathogen defense, either by expression of pathogen-specific bacteriophage or by targeted bursts of reactive oxygen species; (2) vitamin over-expression and delivery; (3) treatment of lactose intolerance, by preferentially metabolizing lactose and funneling it to vitamin production; and (4) regulation of these three treatment functions to produce subpopulations specialized for each function.
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==Why engineer gut microbes?==
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See our wiki at [http://openwetware.org/wiki/IGEM:Caltech/2008 OpenWetWare]
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===The large intestine: an ideal bioreactor===
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[[Image:Digestive system diagram en.svg.png|thumb|200px|right|The human digestive tract]]
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The human intestinal track is a perfect environment for bacteria. It is a 37°C mobile incubator with a constant stream of food. While bacteria are present in all parts of the intestinal track downstream of the stomach, the majority of those bacteria reside in the large intestine. There are approximately 10<sup>12</sup> bacterial per mL in the large intestinal lumen, composed of between 500-1000 different species of bacteria. Of these species, approximately 30 species comprise 99% of all bacteria in the large intestine. Estimating there is roughly 100 mL of feces in the large intestine, all the bacteria in our gut outnumber all the cells in the human body 100 to 1<sup>1</sup>.
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===Probiotic bacteria and other natural examples===
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==[[/Project/Vitamins|Vitamin Production]]==
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[[Image:Lacbr.jpg|thumb|left|Electron micrograph of ''Lactobacillus brevis'', a probiotic lactic acid bacterium]]
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[[Image:Folate_foods.jpg|thumb|left|Tasty foods containing folate]]
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Most of the bacteria in our gut have yet to be characterized because they are difficult to culture, owing to their sensitivity to oxygen. However, several species are known. Many bacterial laboratory strains are derived from the well-known ''Escherichia coli'' (a non-pathogenic type), which is normally present in the large intestine. Because of its use in research, ''E. coli'' is the most well characterized bacteria to date. Another bacteria, ''Bacteroides fragilis'', plays an important role in proper development of the immune system and in controlling intestinal inflammation. Specifically, ''B. fragilis'' produces a starch called polysacharride A. In mice that had been raised in a sterile environment since birth (the intestinal track is initially sterile at birth and requires outside sources of bacteria to populate it), the immune system  had less than normal levels of CD4+ killer T cells, a necessary white blood cell to battle infections. However, the CD4+ levels returned to normal when the mice were raised again in a sterile environment, except for the presence of ''B. fragilis''. Polysaccharide A alone, not the mere presence of Bacteroides fragilis, was responsible for the improvement, since mice raised with ''B. fragilis'' that could not produce polysaccharide A showed the same levels of CD4+ cell as the sterile mice<sup>2</sup>.
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Folate  is the generic term for the various forms of Vitamin B9, which include dihydrofolate (DHF), tetrahydrofolate (THF), and folic acid. An essential vitamin for cell survival, folate is involved in amino synthesis (and thus DNA synthesis) as well as single-carbon-transfer reactions. Though humans don't produce folate, folate deficiency can cause serious birth defects and anemia. As a result, most cereals and breads are supplemented with folate. Folate has also been shown to have good bioavailability in the large intestine, and so is more easily absorbed by the host than other vitamins.  
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Our aim is to engineer a strain of ''E. coli'' that will overexpress folate such that a person without access to green, leafy vegetables or folate-supplemented foods can still obtain the necessary daily amount by having this strain residing in their gut.
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Several bacterial species can cause disease in humans by infecting the gut. ''E. coli'' is commonly associated with food poisoning. ''Salmonela enterica'' is responsible for typhoid fever. ''Campylobacter jejuni'' and ''Shigella'' can cause bowl inflammation, diarrhea and dysentery. Cholera is caused by ''Vibrio cholerae'', which infected around 230,000 people and caused 6,300 deaths in 2006, according to the World Health Organization (WHO)[http://www.who.int/wer/2007/wer8231.pdf]. These pathogens typically cause illness in otherwise healthy people. There are other, more opportunistic bacteria, which infect people in hospital settings who are otherwise sick or undergoing treatment that puts them at greater risk for infection of their intestinal track. Paradoxically, the treatment of one pathogen with antibiotics can make that same patient more susceptible to infection of their gut by opportunistic pathogens. It is thought that the right balance of natural gut flora prevents these opportunistic pathogens from colonizing the colon<sup>3</sup>.
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==[[/Project/Lactose intolerance|Lactose intolerance]]==
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As a further example, humans cannot produce vitamin K, an important cofactor in blood clotting. Instead, the vitamin is provided by various species of the gut microbiota, which collectively produce more than 30 times the vitamin K recommended daily allowance<sup>4</sup>.
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Lactose intolerance is the inability to metabolize lactose in the small intestine. For a non lactose intolerant person, he or she is able to express β-galactosidase in the small intestine which cleaves lactose into glucose and galactose. Glucose and galactose are then taken up by the host. For a lactose intolerant person, lactose travels passed the small intestine into the large intestine where it is metabolized by the gut flora. The osmolarity changes in the large intestine due to the large amounts of sugar, pulling more water into the large intestine. Due to this rush of water, lactose intolerant persons may experience diarrhea. Lactose is metabolized in this order: Lactose --> glucose + galactose --> lactic acid + acetic acid --> H<sub>2</sub> --> methane. A different bacteria is involved in each step, so we can't engineer our strain to metabolize lactose, because it will eventually get metabolized into methane, which is the cause of abdominal pain and unwanted gas.
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Instead, we will engineer a strain of ''E. coli'' that will express the β-Gal gene in the large intestine to quickly cleave lactose. The host will then be able to reabsorb the glucose and galactose from the large intestine.
 
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==[[/Project/Oxidative Burst|Oxidative Burst]]==
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===Nissle 1917: Probiotic, commercially available ''E. coli''===
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[[Image:neutrophil-shigella.jpg|thumb|left|A neutrophil trapping Shigella]]
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[[Image:packshot_mutaflor.jpg|thumb|right|Mutaflor - a commercially available preparation of Nissle 1917]]
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Several types of bacteria can cause illness in humans by infecting the gut. ''Salmonella'' and ''E. coli'' are probably the two people most frequently associate with food poisoning. There are other pathogenic bacteria that can infect our gut as well. ''Shigella'' and ''Campylobacter'' can cause cramping, diarrhea and dysentery. ''Vibrio cholerae'', which also infects the gut, is the cause of cholera. The body normally relies on white blood cells (neutrophils) to clear bacteria from the body. Once a bacterium is engulfed, the neutrophil releases a sudden and toxic amount of reactive oxygen species, comprised of a mixture of superoxide, hydrogen peroxide, and hypochlorous acid. This is event is termed the oxidative burst. However, white blood cells do not patrol the gut lumen and so there is no active clearance of pathogens. The goal of this project is to engineer a beneficial gut microbe capable of detecting a harmful bacterium and, in turn, generate an oxidative burst sufficient to kill the pathogen.
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Nissle 1917 is a commercially available[http://www.ardeypharm.de/en/] non-pathogenic, probiotic strain of ''E. coli''. It has been successfully used to treat gastrointestinal disorders including colitis and intestinal bowel disease<sup>5</sup> and shows little immunostimulatory activity<sup>6</sup>. Engineered versions of the Nissle 1917 strain have been developed as anti-HIV<sup>7</sup> and anti-cholera<sup>8</sup> agents. The ability of Nissle 1917 to efficiently colonize the gut without provoking an inflammatory response makes it an ideal chassis for ''in situ'' applications in biomedicine and biotechnology.
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==[[/Project/Phage Pathogen Defense|Phage Pathogen Defense]]==
 
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[[Image:Phage.jpg|thumb|left|Phage attached to bacteria, electron micrograph]]
 
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Another aspect of bacterial pathogen defense for our probiotic is to produce bacteriophages, which would rapidly infect and wipe out all of the pathogens. There are basically methods to approach phage production, differentiated by the type of phage used. The first uses the bacteriophage λ, which targets E. Coli. The other is exploring the use of a temperate bacteriophage from B. Subtilis, however this method, if successful, can be adapted to temperate bacteriophages of any bacterial strain.
 
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Bacteriophage λ is a temperate phage with an E. Coli. host, λ infects E. Coli through the lamB receptor, and absence of this receptor prevents λ infection. We will takes advantage of this aspect of bacteriophage λ to create E. Coli which are resistant to the phage, but release the phage to destroy susceptible pathogenic E. Coli.
 
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The second approach is more versatile, and can target more strains of pathogenic bacteria. The goal is to create a phasmid out of the genome of a temperate bacteriophage. A phasmid combines a E. Coli plasmid Origin of Replication with a linear phage genome, circularizing it. This allows the phasmid to pass on as a plasmid within E. Coli, however when transferred to its native host, the phage phage is induced.
 
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==[[/Project/Population Variation|Population Variation]]==
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For more details, please see our [[Team:Caltech/Project|<font style="color:#BB4400">project</font>]] page.
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Given that we have created four different states for a cell to be in, we need to in some manner combine them into one system. However, we need to ensure that any particular cell is in only one of these states, and not more than one, or else the load on the cell may be too big. In other words, we want to make these states mutually exclusive.
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To do so, two new devices have been created: a randomly activated off-to-on switch and a population variation generator. Initially, a cell is in a default state (S0), the switch is off, and the fate of the cell is undetermined. However, each time a cell replicates the plasmid that contains the switch, there is a chance that the switch turns on. Once the switch is on, it activates the population variation generator, which in turn determines the fate of the cell by setting it to one of three states - S1, S2, or S0 (the original default). That cell and all of its descents then stay in the determined state.  
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===References===
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# Hooper LV, Midtvedt T, and Gordon JI. '''How host-microbial interactions shape the nutrient environment of the mammalian intestine'''. ''Annu Rev Nutr'' 2002; 22 283-307.
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#  Mazmanian SK, Liu CH, Tzianabos AO, and Kasper DL. '''An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system'''. ''Cell'' 2005 Jul 15; 122(1) 107-18.
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# Donskey CJ. '''The role of the intestinal tract as a reservoir and source for transmission of nosocomial pathogens'''. ''Clin Infect Dis'' 2004 Jul 15; 39(2) 219-26.
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# Suttie JW. '''The importance of menaquinones in human nutrition'''. ''Annu Rev Nutr'' 1995; 15 399-417.
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# Krammer HJ, Kamper H, von Bunau R, Zieseniss E, Stange C, Schlieger F, Clever I, and Schulze J. '''Probiotic drug therapy with E. coli strain Nissle 1917 (EcN): results of a prospective study of the records of 3,807 patients'''. ''Z Gastroenterol'' 2006 Aug; 44(8) 651-6.
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# Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, Buer J, and Bruder D. '''Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules'''. ''FEMS Immunol Med Microbiol'' 2005 Mar 1; 43(3) 373-84.
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# Rao S, Hu S, McHugh L, Lueders K, Henry K, Zhao Q, Fekete RA, Kar S, Adhya S, and Hamer DH. '''Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide'''. ''Proc Natl Acad Sci U S A'' 2005 Aug 23; 102(34) 11993-8.
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# Duan F and March JC. '''Interrupting Vibrio cholerae infection of human epithelial cells with engineered commensal bacterial signaling'''. ''Biotechnol Bioeng'' 2008 Sep 1; 101(1) 128-34.
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}}

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Engineering multi-functional probiotic bacteria

Engineered gut flora

The human gut houses a diverse collection of microorganisms, with important implications for the health and welfare of the host. We aim to engineer a member of this microbial community to provide innovative medical treatments. Our work focuses on four main areas: (1) pathogen defense either by expression of pathogen-specific bacteriophage or by targeted bursts of reactive oxygen species; (2) prevention of birth defects by folate over-expression and delivery; (3) treatment of lactose intolerance by cleaving lactose to allow absorption in the large intestine; and (4) regulation of these three treatment functions to produce renewable subpopulations specialized for each function. Our research demonstrates that synthetic biology techniques can be used to modify naturally occurring microbial communities for applications in biomedicine and biotechnology.

Why engineer gut microbes?

The large intestine: an ideal bioreactor

The human digestive tract

The human intestinal track is a perfect environment for bacteria. It is a 37°C mobile incubator with a constant stream of food. While bacteria are present in all parts of the intestinal track downstream of the stomach, the majority of those bacteria reside in the large intestine. There are approximately 1012 bacterial per mL in the large intestinal lumen, composed of between 500-1000 different species of bacteria. Of these species, approximately 30 species comprise 99% of all bacteria in the large intestine. Estimating there is roughly 100 mL of feces in the large intestine, all the bacteria in our gut outnumber all the cells in the human body 100 to 11.

Probiotic bacteria and other natural examples

Electron micrograph of Lactobacillus brevis, a probiotic lactic acid bacterium

Most of the bacteria in our gut have yet to be characterized because they are difficult to culture, owing to their sensitivity to oxygen. However, several species are known. Many bacterial laboratory strains are derived from the well-known Escherichia coli (a non-pathogenic type), which is normally present in the large intestine. Because of its use in research, E. coli is the most well characterized bacteria to date. Another bacteria, Bacteroides fragilis, plays an important role in proper development of the immune system and in controlling intestinal inflammation. Specifically, B. fragilis produces a starch called polysacharride A. In mice that had been raised in a sterile environment since birth (the intestinal track is initially sterile at birth and requires outside sources of bacteria to populate it), the immune system had less than normal levels of CD4+ killer T cells, a necessary white blood cell to battle infections. However, the CD4+ levels returned to normal when the mice were raised again in a sterile environment, except for the presence of B. fragilis. Polysaccharide A alone, not the mere presence of Bacteroides fragilis, was responsible for the improvement, since mice raised with B. fragilis that could not produce polysaccharide A showed the same levels of CD4+ cell as the sterile mice2.

Several bacterial species can cause disease in humans by infecting the gut. E. coli is commonly associated with food poisoning. Salmonela enterica is responsible for typhoid fever. Campylobacter jejuni and Shigella can cause bowl inflammation, diarrhea and dysentery. Cholera is caused by Vibrio cholerae, which infected around 230,000 people and caused 6,300 deaths in 2006, according to the World Health Organization (WHO)[1]. These pathogens typically cause illness in otherwise healthy people. There are other, more opportunistic bacteria, which infect people in hospital settings who are otherwise sick or undergoing treatment that puts them at greater risk for infection of their intestinal track. Paradoxically, the treatment of one pathogen with antibiotics can make that same patient more susceptible to infection of their gut by opportunistic pathogens. It is thought that the right balance of natural gut flora prevents these opportunistic pathogens from colonizing the colon3.

As a further example, humans cannot produce vitamin K, an important cofactor in blood clotting. Instead, the vitamin is provided by various species of the gut microbiota, which collectively produce more than 30 times the vitamin K recommended daily allowance4.


Nissle 1917: Probiotic, commercially available E. coli

Mutaflor - a commercially available preparation of Nissle 1917

Nissle 1917 is a commercially available[2] non-pathogenic, probiotic strain of E. coli. It has been successfully used to treat gastrointestinal disorders including colitis and intestinal bowel disease5 and shows little immunostimulatory activity6. Engineered versions of the Nissle 1917 strain have been developed as anti-HIV7 and anti-cholera8 agents. The ability of Nissle 1917 to efficiently colonize the gut without provoking an inflammatory response makes it an ideal chassis for in situ applications in biomedicine and biotechnology.

For more details, please see our project page.

References

  1. Hooper LV, Midtvedt T, and Gordon JI. How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annu Rev Nutr 2002; 22 283-307.
  2. Mazmanian SK, Liu CH, Tzianabos AO, and Kasper DL. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 2005 Jul 15; 122(1) 107-18.
  3. Donskey CJ. The role of the intestinal tract as a reservoir and source for transmission of nosocomial pathogens. Clin Infect Dis 2004 Jul 15; 39(2) 219-26.
  4. Suttie JW. The importance of menaquinones in human nutrition. Annu Rev Nutr 1995; 15 399-417.
  5. Krammer HJ, Kamper H, von Bunau R, Zieseniss E, Stange C, Schlieger F, Clever I, and Schulze J. Probiotic drug therapy with E. coli strain Nissle 1917 (EcN): results of a prospective study of the records of 3,807 patients. Z Gastroenterol 2006 Aug; 44(8) 651-6.
  6. Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, Buer J, and Bruder D. Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules. FEMS Immunol Med Microbiol 2005 Mar 1; 43(3) 373-84.
  7. Rao S, Hu S, McHugh L, Lueders K, Henry K, Zhao Q, Fekete RA, Kar S, Adhya S, and Hamer DH. Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide. Proc Natl Acad Sci U S A 2005 Aug 23; 102(34) 11993-8.
  8. Duan F and March JC. Interrupting Vibrio cholerae infection of human epithelial cells with engineered commensal bacterial signaling. Biotechnol Bioeng 2008 Sep 1; 101(1) 128-34.
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