Team:Saarland/Test

From 2014.igem.org

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<h1> 1. Search and Find Anti-Cancer Genes  </h1>
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<h1> 3. Find allies and join forces </h1>
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<h2>The naked mole rat (<i>Heterocephalus glaber</i>)</h2>
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<h2><i>B. megaterium </i> </h2>  
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Living strictly in subterranean colonies in the semiarid regions of East Africa including parts of Kenya, Ethiopia and Somalia, the naked mole rat is the only known eusocial mammal (Honeycutt et al. 1991). A single breeding female individual, also referred as queen, maintains her subordinates in a reproductively quiescent state. The behavioural division of labor can be compared to that of bee colonies (Jarvis, 1981).
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Due to the extreme constraints of its natural subterranean habitat including full darkness, low oxygen and high carbon dioxide concentrations, the naked mole rat adapted striking physiological features that distinguish it from other rodents. Based on the average body weight centering around 35 g, naked mole rats remarkably exceed their theoretical life span of 10 years, since captive individuals of 30 years could frequently be observed (Buffenstein and Jarvis, 2002). Compared to the life span of other similarly sized rodents such as mice, the life span of a naked mole rat is 9 times higher. Furthermore naked mole rats show a delayed ageing process mainly resulting from a characteristic low metabolic and respiratory rate. The latter probably evolved as an adaption to the challenging food acquisition and the life in the oxygen poor environment (Buffenstein, 2005). The delayed aging process, also called negligible senescence, offers an interesting opportunity for studies on age related research (Buffenstein, 2008).
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Another outstanding feature of the naked mole rat is its unusual resistance to both spontaneous cancer and experimentally induced carcinogenesis (Liang et al. 2010, Selunov et al. 2009). The cause of this cancer resistance has recently been identified by Tian et al. (2013). It is attributed to the secretion of an extremely high molecular mass hyaluronic acid (HMM-HA) in naked mole rat fibroblasts. HMM-HA is playing a central role in our project <br>
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<i>B. megaterium </i> belongs to the group of Gram-positive, aerobic bacteria. It’s typically found in soil, but can also survive in sea water or dried foods (Vary <i>et al</i>., 2007). Since its discovery in 1884, it has become one of the most important organisms for industrial and research purposes, because it combines numerous useful characteristics. Due to its extraordinary size of up to 1,5 µm x 4 µm and it`s impressive cell volume of more than 60 µm3, <i>B. megaterium</i>is predestined for studies regarding cell structure and protein localisation (Boyke <i>et al</i>., 2010; Vary <i>et al</i>., 1992). Furthermore it is able to metabolise more than 62 carbon sources, making its cultivation efficient and inexpensive (Millet <i>et al</i>., 1962). Probably the most useful characteristic for our purposes is its ability of preserving plasmids over multiple generations, making it a good specimen for the secretion of proteins and other organic molecules. Especially concerning the <i>B. megaterium</i> strain MS941 that was derived from the strain DSM319 by knockout of the neutral protease gene nprM (Wittchen <i>et a/i>l. 1995). For this reason the non sporulating strain MS941 is ideal for heterologous protein expression. This was shown by overexpression of GFP (Stammen <i>et a</i>l., 2007).<br> <br>  
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(you can read more about this unique molecule <b>HERE </b> )
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Missing the neuropeptide Substance P in the skin the naked mole rat additionally features a reduced sense of pain (Park et al., 2008). All together the naked mole rat can be referred as an uncommon model organism in scientific research, although it is in possession of several promising traits that can lead to a more profound understanding of important biological and biomedical questions. <br>
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<h2> Properties and Synthesis of hyaluronic acid </h2>
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Additionally, the biosynthetic pathway of hyaluronic acid precursor molecules is already established in <i>B. megaterium</i> since UDP-N-acetyl-D-glucosamine and UDP-D-glucuronic acid are also essential components for cell wall synthesis in Gram-positive bacteria. Manual supplementation of these extremely expensive precursor molecules is not longer necessary. This could contribute to a future profitable biotechnological production of the high molecular mass hyaluronic acid (HMM-HA) of the naked mole rat. Homology searches in the <i>B. megaterium</i> genome have also shown that there are no endogenous hyaluronidases, which would otherwise immediately degrade the produced hyaluronic acid. Furthermore <i>B. megaterium</i> does not possess an endogenous hyaluronan synthase (Has). For this reason our team can be sure that hyaluronic acid has exclusively been synthesised by the correct enzyme and features the correct molecular weight and anti carcinogenic properties.<br> <br>
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Hyaluronic acid (HA) is a linear polysaccharide that is naturally occurring in various living organisms. The macromolecular chain consists of repeating disaccharide units of alternating D-glucuronic acid and N-acetyl-D-glucosamine and can reach variable chain lengths of up to 20.000 disaccharide units resulting in molecular weights of up to 107 Da. <br>
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For the optimisation of hyaluronic acid production our team intends to overexpress endogenous <i>B. megaterium</i> proteins that are necessary for the production of hyaluronic acid precursor molecules. We think that the flow equilibrium of metabolites will be shifted towards production of HA, rather than to cell wall components. This approach could make a considerable contribution for high yield production of HMM-HA. The identification of corresponding genes in </i>B. megaterium</i> is based on the biosynthetic pathway for the HA production in group A and group C streptococci as well as for HA production in <i>B. subtilis</i> (Widner <i>et al</i>. 2005). The proposed biosynthetic pathway for production of the HA precursor molecules in <i>B. megaterium</i> after <i>in silico</i> gene homology search on MegaBac v9 platform is shown in figure 2.
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HA is synthesised by the integral membrane protein hyaluronan synthase (Has). There are three different hyaluronan synthases known in vertebrates which are Has1, Has2 and Has3 (Necas, 2008). The enzyme catalyzes the linkage of UDP-D-glucuronic acid and UDP-N-acetyl-D-glucosamine to the long, linear HA chain (Garg and Hales, 2004). The energy for this process is delivered by the activated carbohydrate UDP which is used as a substrate by the hyaluronan synthases. The synthesis of the HA itself is a highly regulated process. Hyaluronidases (HAase) play also a crucial role in the regulation of HA amounts as they consequently lower the HA concentration in tissues (Necas, 2008).<br>
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As a main component of the pericellular matrix and extracellular matrix HA is ubiquitously found in human and animal connective tissues with significant amounts in skin, umbilical cord, synovial fluid, and vitreous humor. High concentrations of HA are also found in lung, kidney, brain, and muscle tissues (Garg and Hales, 2004; Necas, 2008). Due to its unique properties concerning viscoelasticity and excellent moisture retention capacity, HA is functionally involved in many biological processes mainly including hydration of tissues and lubrication of moveable parts of the body such as intervertebral discs and joints. These exceptional physiologic properties along with its high biocompatibility and non-immunogenicity have contributed to the fact that HA has found success in an extraordinarily broad range of biomedical and cosmetic applications. Local intra articular injections of HA in patients suffering from osteoarthritis for example restore the shock absorbance, reduce the degeneration of cartilage and lead to an alleviation of the painful symptoms in affected joints (Uthman et al., 2003). The glycosaminoglycane is additionally used in medicine to faciliate wound healing and dermal regeneration in skin cosmetics (Necas, 2008). Apart from this a receptor-mediated role in gene expression, proliferation, migration, tumor development and inflammation has been described (Turley et al., 2002; Toole et al., 2002; Hascall et al., 2004).<br> <br>
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However, when the HA will be successfully produced as we described in figure 2, then it is supposed to be secreted into the medium. Since studies with the similar<i> B. subtilis</i> expressing a streptococci hyaluronan synthase (HasA) have already shown similar results. It’s either possible that there is a specific transporter for oligosaccharides mediating HA secretion into the medium, or that the multi membrane domains of the hyaluronan synthase themselves form a channel for the secretion of the nascent HA chain (Weigel, 2002)
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<h2> The high molecular mass hyaluronic acid of the naked mole rat</h2>
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The high molecular mass hyaluronic acid (HMM-HA) of the naked mole rat shows the same basic structure composed of a repetitive disaccharide unit, built up of D-glucuronic acid and N-acetyl-D-glucosamine, compared to the human HA. However, the HMM-HA has a molecular weight of 6-12 MDa which is significantly higher than the human pendant ranging between 0,5 and 2 MDa. <br>
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The Has of the naked mole rat is in possession of 7 putative transmembrane domains and one large cytoplasmic loop which is supposed to be the catalytic center for HA Synthesis.
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In contrast to the highly conserved catalytic domain of hyaluronan synthases in other vertebrates the naked mole rats HA synthase features two unique changes in the amino acid sequence as two asparagines have been substituted by serines. This might be the cause for the raised activity of the naked mole rat´s Has. <br>
 
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In the recently published paper of Tian et al. (2013) the exceptionally HMM-HA of the naked mole rat is postulated to mediate the cancer resistance of the naked mole rat. In fact the resistance seems to be a side effect of the evolutionary adaptation. Thereby the anti carcinogenic effect is based on the hypersensitivity of contact inhibition, also referred as early contact inhibition (ECI) (Seluanov et al., 2009). Contact inhibition is a cellular mechanism inducing cell cycle arrest and consequently disabling cell divisions in order to counteract uncontrolled cell proliferation during tumor formation. A proposed signaling pathway for induction of ECI by HMM-HA is shown in Figure 3. <br>
 
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An evidence for the induction of ECI by HMM-HA was established by adding HAse to naked mole rat fibroblasts leading to reverse the effect of early contact inhibition. Moreover a decrease of the HMM-HA concentration in naked mole rat cells caused by knockdown of Has2 or overexpression of hyaluronidase combined with the simultaneous expression of the viral oncoprotein SV40 LT and H-Ras V12 in xenograft transplantations in mice showed increased tumor formation (Tian et al. 2013).
 
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At this point the effect of HMM-HA on human cells has not been tested since the HMM-HA is not commercially available yet. The biotechnological production of the HMM-HA with its anti carcinogenic properties offers a promising approach for the future fight against cancer. Therefore the HMM-HA production is the primary objective of our iGEM project.
 
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Revision as of 11:18, 9 October 2014



3. Find allies and join forces


B. megaterium


B. megaterium belongs to the group of Gram-positive, aerobic bacteria. It’s typically found in soil, but can also survive in sea water or dried foods (Vary et al., 2007). Since its discovery in 1884, it has become one of the most important organisms for industrial and research purposes, because it combines numerous useful characteristics. Due to its extraordinary size of up to 1,5 µm x 4 µm and it`s impressive cell volume of more than 60 µm3, B. megateriumis predestined for studies regarding cell structure and protein localisation (Boyke et al., 2010; Vary et al., 1992). Furthermore it is able to metabolise more than 62 carbon sources, making its cultivation efficient and inexpensive (Millet et al., 1962). Probably the most useful characteristic for our purposes is its ability of preserving plasmids over multiple generations, making it a good specimen for the secretion of proteins and other organic molecules. Especially concerning the B. megaterium strain MS941 that was derived from the strain DSM319 by knockout of the neutral protease gene nprM (Wittchen et a/i>l. 1995). For this reason the non sporulating strain MS941 is ideal for heterologous protein expression. This was shown by overexpression of GFP (Stammen <i>et al., 2007).

Additionally, the biosynthetic pathway of hyaluronic acid precursor molecules is already established in B. megaterium since UDP-N-acetyl-D-glucosamine and UDP-D-glucuronic acid are also essential components for cell wall synthesis in Gram-positive bacteria. Manual supplementation of these extremely expensive precursor molecules is not longer necessary. This could contribute to a future profitable biotechnological production of the high molecular mass hyaluronic acid (HMM-HA) of the naked mole rat. Homology searches in the B. megaterium genome have also shown that there are no endogenous hyaluronidases, which would otherwise immediately degrade the produced hyaluronic acid. Furthermore B. megaterium does not possess an endogenous hyaluronan synthase (Has). For this reason our team can be sure that hyaluronic acid has exclusively been synthesised by the correct enzyme and features the correct molecular weight and anti carcinogenic properties.

For the optimisation of hyaluronic acid production our team intends to overexpress endogenous B. megaterium proteins that are necessary for the production of hyaluronic acid precursor molecules. We think that the flow equilibrium of metabolites will be shifted towards production of HA, rather than to cell wall components. This approach could make a considerable contribution for high yield production of HMM-HA. The identification of corresponding genes in </i>B. megaterium</i> is based on the biosynthetic pathway for the HA production in group A and group C streptococci as well as for HA production in B. subtilis (Widner et al. 2005). The proposed biosynthetic pathway for production of the HA precursor molecules in B. megaterium after in silico gene homology search on MegaBac v9 platform is shown in figure 2.

However, when the HA will be successfully produced as we described in figure 2, then it is supposed to be secreted into the medium. Since studies with the similar B. subtilis expressing a streptococci hyaluronan synthase (HasA) have already shown similar results. It’s either possible that there is a specific transporter for oligosaccharides mediating HA secretion into the medium, or that the multi membrane domains of the hyaluronan synthase themselves form a channel for the secretion of the nascent HA chain (Weigel, 2002)


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