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                     <p>Your place:&nbsp;<a href="https://2015.igem.org/Team:Nankai">Home</a>&nbsp;&gt;&nbsp;<a href="https://2015.igem.org/Team:Nankai/Parts">Parts</a>&nbsp;&gt;&nbsp;<a href="https://2015.igem.org/Team:Nankai/Composite_Part">Composite Parts</a></p>
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                     <p>Your place:&nbsp;<a href="https://2015.igem.org/Team:Nankai">Home</a>&nbsp;&gt;&nbsp;<a href="https://2015.igem.org/Team:Nankai/Parts">Parts</a>&nbsp;&gt;&nbsp;<a href="https://2015.igem.org/Team:Nankai/Composite_Part">Composite Part</a></p>
 
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<h4>What is γ-PGA?</h4>
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<h4>Composite Parts</h4>
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<p>Poly-γ-glutamic acid (γ-PGA) is an important, naturally occurring polyamide consisting of D/L-glutamate monomers. Unlike typical peptide linkages, the amide linkages in γ-PGA are formed between the α-amino group and the γ-carboxyl group. γ-PGA exhibits many favorable features such as biodegradable, water soluble, edible and non-toxic to humans and the environment. Therefore, it has been widely used in fields of foods, medicines, cosmetics and agriculture and many unique applications, such as a sustained release material and drug carrier, curable biological adhesive, biodegradable fibres, and highly water absorbable hydrogels.</p>
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<p style="position: relative; top: 0px; left: 20px; width: 700px; font-size: 18px; font-family: calibri,Arial, Helvetica, sans-serif; text-align: justify; line-height: 30px;">Promoter P<sub>xyl</sub> together with promoter P<sub>lacI</sub>, repressor LacI (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1628201">BBa_K1628201</a>), promoter P<sub>grac</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1628202">BBa_K1628202</a>) and repressor XylR (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1628203">BBa_K1628203</a>) formed a metabolic toggle switch.  Promoter P<sub>xyl</sub>(<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1628002">BBa_K1628002</a>) is a promoter of xylose operon regulate by repressor XylR. Promoter P<sub>lacI</sub> is a promoter of lactose operon and LacI is a repressor of lactose operon regulating promoter P<sub>lacI</sub>. PlacI is the native promoter of LacI. Promoter P<sub>grac</sub> is a promoter of lactose operon regulated by repressor LacI. </p>
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<h4>How can we produce it?</h4>
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<p style="position: relative; top: 0px; left: 20px; width: 700px; font-size: 18px; font-family: calibri,Arial, Helvetica, sans-serif; text-align: justify; line-height: 30px;">We used this device to regulate the expression of <em>odhAB</em> genes in <em>B. amyloliquefaciens</em> NK-1 (showed in Figure 1). Without IPTG, the promoter P<sub>grac</sub> is inhibited by suppressor LacI and the supreessor XylR will not synthesized, thus the promoter P<sub>xyl</sub> is active and <em>odhAB</em> genes are expressed. When IPTG is added, the<em> xylR</em> gene is expressed and the suppressor XylR is synthesized thereafter inhibited the expression of <em>odhAB</em> genes.</p>
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<p>Strains capable for producing γ-PGA are divided into two categories based on their requirement for glutamate acid: glutamate-dependent strains and glutamate-independent strains. Glutamate-independent strains are preferable for industrial production because of their low cost and simplified fermentation process. However, compared with glutamate-dependent strains, their lower γ-PGA productivity limits their industrial application.Therefore, the construction of a glutamate-independent strain with high γ-PGA yield is important for industrial applications.</p>
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<h4>Who can produce it?</h4>
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<h6>Figure 1. Metabolic toggle switch to regulate the expression of <em>odhAB</em></h6>
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<p>Bacillusamyloliquefaciens LL3, isolated from fermented food, is a glutamate-independent strain, which can produce 3-4 g/L γ-PGA with sucrose as its carbon source and ammonium sulfate as its nitrogen source. The B. amyloliquefaciens LL3 strain was deposited in the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 208109 and its whole genome has been sequenced in 2011. In this study, we aimed to improve the γ-PGA production based on the B. amyloliquefaciens NK-1 strain (a derivative of LL3 strain with its endogenous plasmid and upp gene deleted).</p>
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<p style="position: relative; top: 0px; left: 20px; width: 700px; font-size: 18px; font-family: calibri,Arial, Helvetica, sans-serif; text-align: justify; line-height: 30px;">We transformed the plasmids pHT01-<em>xylR</em> and pCB-Pxyl into the NK-1 strain, to verify the activity of metabolic toggle switch (see it on our wiki).  Fresh colonies of <em>Bacillus amyloliquefaciens</em> strains (NK-1 strain containing plasmids pHT01-xylR and pCB-Pxyl and the control NK-1 strain containing plasmids pHT01 and pCB-Pxyl) were first cultured  overnight in test tubes containing 5 mL LB liquid and then inoculated into 100  mL fresh fermentation medium. We added 1mM IPTG into the medium after 12h of cultivation. The β-galactosidase activity were measured at 12h, 18h, 24h, 30h,  36h, 42h to test the effect of metabolic toggle switch on the expression of <em>bgaB</em>.<br>
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<h4>What did we do?</h4>
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As shown in Figure 2,  β-galactosidase enzyme activity dropped considerably after 30 hours of fermentation. The inhibited expression of <em>bgsB </em>in experiment group (NPP+IPTG) indicated that the metabolic toggle switch  we constructed is functional in <em>B.  amyloliquefaciens</em> NK-1 strain.</p>
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<p>In order to improve γ-PGA production, we employed two strategies to fine-tune the synthetic pathways and balance the metabolism in the glutamate-independent B. amyloliquefaciens NK-1 strain. Firstly, we constructed a metabolic toggle switch in the NK-1 strain to inhibit the expression of ODHC (2-oxoglutarate dehydrogenase complex) by adding IPTG in the stationary stage and distribute the metabolic flux more frequently to be used for γ-PGA precursor-glutamate synthesis. As scientists had found that the activity of ODHC was rather low when glutamate was highly produced in a Corynebacterium glutamicum strain. Second, to balance the increase of endogenous glutamate production, we optimized the expression level of pgsBCA genes (responsible for γ-PGA synthesis) by replacing its native promoter to seven different strength of promoters. Through these two strategies, we aimed to obtain a γ-PGA production improved mutant strain.<a href="https://2015.igem.org/Team:Nankai/Experiments">Click for more detail.</a></p>
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<img src="https://static.igem.org/mediawiki/2015/2/26/Parts_figure_switch_function_verification.jpeg">
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<h6>Figure 2. Verification of the metabolic toggle switch’s function. IPTG was added to the medium after 12h cultivation.</h6>
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<h6><a href="https://2015.igem.org/Team:Nankai/Basic_Part">Basic Parts</a></h6>
 
<h6><a href="https://2015.igem.org/Team:Nankai/Basic_Part">Basic Parts</a></h6>
 
<h6><a href="https://2015.igem.org/Team:Nankai/Composite_Part">Composite Parts</a></h6>
 
<h6><a href="https://2015.igem.org/Team:Nankai/Composite_Part">Composite Parts</a></h6>
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<h6><a href="https://2015.igem.org/Team:Nankai/Part_Collection">Part Collection</a></h6>
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<img src="https://static.igem.org/mediawiki/2015/d/de/Partsfigure_new1.jpeg">
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                                                 <p>Preparing for LB medium.</p>
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                                                 <p>&nbsp;</p>
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<img src="https://static.igem.org/mediawiki/2015/4/40/Partsfigure_new2.jpeg">
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                                                <p>Cultured LL3.</p>
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                      <p>&nbsp;</p>
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                                                <p>In the progress of fermentation.</p>
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                      <p>&nbsp;</p>
  
 
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<h6>References</h6>
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<p>1. Ashiuchi, M., Misono, H., 2002. Biochemistry and molecular genetics of poly-γ-glutamate synthesis. Appl. Biochem. Biotechnol. 59, 9–14.</br>
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2. Kunioka, M., 1997. Biosynthesis and chemical reactions of poly(amino acid)s from
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microorganisms. Appl. Microbiol. Biotechnol. 47, 469–475.</br>
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3. Shih, I.L., Van, Y.T., 2001. The production of poly(γ-glutamic acid) from microorganism and its various applications. Bioresour. Technol. 79, 207–225.</br>
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4. Li, C., 2002. Poly(L-glutamic acid)--anticancer drug conjugates. Adv. Drug Deliver. Rev. 54, 695–713.</br>
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5. Liang, H.F., Chen, C.T., Chen, S.C., Kulkarni, A.R., Chiu, Y.L., Chen, M.C., Sung, H.W., 2006. Paclitaxel-loaded poly(γ-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system for the treatment of liver cancer. Biomaterials. 27, 2051–2059.</br>
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6. Richard, A., Margaritis, A., 2001. Poly (glutamic acid) for biomedical applications. Crit. Rev. Biotechnol. 21, 219–232.</br>
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7. Park, Y.J., Liang, J., Yang, Z., Yang, V.C., 2001. Controlled release of clot-dissolving tissue-type plasmmogen activator from a poly(L-glutamic acid) semi-interpenetrating polymer network hydrogel. J. Control. Release. 74, 243–247.</br>
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8. Cao, M.F., Geng, W.T., Liu, L., Song, C.J., Xie, H., Guo, W.B., Jin, Y.H., Wang, S.F., 2011. Glutamic acid independent production of poly-γ-glutamic acid by Bacillus amyloliquefaciens LL3 and cloning of pgsBCA genes. Bioresour. Technol. 102, 4251–4257.</br>
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9. Geng, W.T., Cao, M.F., Song, C.J., Xie, H., Liu, L., Yang, C., Feng, J., Zhang, W., Jin, Y.H., Du, Y., Wang, S.F., 2011. Complete genome sequence of Bacillus amyloliquefaciens LL3, which exhibits glutamic acid-independent production of poly-γ-glutamic acid. J. Bacteriol. 193, 3393–3394.</br>
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10. Feng, J., Gao, W.X., Gu, Y.Y., Zhang, W., Cao, M.F., Song, C.J., Zhang, P., Sun, M., Yang, C.,  Wang, S.F., 2014a. Functions of poly-gamma-glutamic acid (γ-PGA) degradation genes in γ-PGA synthesis and cell morphology maintenance. Appl. Microbiol. Biotechnol. 98, 6397–6407.</br>
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11. Uy, D., Delaunay S., Germain, P., Engasser, J.M., Goergen, J.L. 2003. Instability of glutamate production by Corynebacterium glutamicum 2262 in continuous culture using the temperature-triggered process. J. Biotech. 104, 173-184.</p>
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Latest revision as of 15:21, 18 September 2015

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Promoter Pxyl together with promoter PlacI, repressor LacI (BBa_K1628201), promoter Pgrac (BBa_K1628202) and repressor XylR (BBa_K1628203) formed a metabolic toggle switch. Promoter Pxyl(BBa_K1628002) is a promoter of xylose operon regulate by repressor XylR. Promoter PlacI is a promoter of lactose operon and LacI is a repressor of lactose operon regulating promoter PlacI. PlacI is the native promoter of LacI. Promoter Pgrac is a promoter of lactose operon regulated by repressor LacI.

We used this device to regulate the expression of odhAB genes in B. amyloliquefaciens NK-1 (showed in Figure 1). Without IPTG, the promoter Pgrac is inhibited by suppressor LacI and the supreessor XylR will not synthesized, thus the promoter Pxyl is active and odhAB genes are expressed. When IPTG is added, the xylR gene is expressed and the suppressor XylR is synthesized thereafter inhibited the expression of odhAB genes.

Figure 1. Metabolic toggle switch to regulate the expression of odhAB

We transformed the plasmids pHT01-xylR and pCB-Pxyl into the NK-1 strain, to verify the activity of metabolic toggle switch (see it on our wiki). Fresh colonies of Bacillus amyloliquefaciens strains (NK-1 strain containing plasmids pHT01-xylR and pCB-Pxyl and the control NK-1 strain containing plasmids pHT01 and pCB-Pxyl) were first cultured overnight in test tubes containing 5 mL LB liquid and then inoculated into 100 mL fresh fermentation medium. We added 1mM IPTG into the medium after 12h of cultivation. The β-galactosidase activity were measured at 12h, 18h, 24h, 30h, 36h, 42h to test the effect of metabolic toggle switch on the expression of bgaB.
As shown in Figure 2, β-galactosidase enzyme activity dropped considerably after 30 hours of fermentation. The inhibited expression of bgsB in experiment group (NPP+IPTG) indicated that the metabolic toggle switch we constructed is functional in B. amyloliquefaciens NK-1 strain.

Figure 2. Verification of the metabolic toggle switch’s function. IPTG was added to the medium after 12h cultivation.
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