Difference between revisions of "Team:Nankai/Composite Part"

 
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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></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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<h2 class="page-title">Team Parts</h2>
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<h2 class="page-title">Composite Parts</h2>
 
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<h4>Composite Parts</h4>
 
<h4>Composite Parts</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;"><b>Composite part example 1 </b></br>
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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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  （consists of basic part1 and basic part2）<a href="http://partsregistry.org/Part:BBa_K1031221">BBa_K1031221</a>, <a href="http://partsregistry.org/Part:BBa_K1031222">BBa_K1031222</a>, <a href="http://partsregistry.org/Part:BBa_K1031223">BBa_K1031223</a> and <a href="http://partsregistry.org/Part:BBa_K1031224">BBa_K1031224</a> are the DmpR biosensors using different RBS preceding sfGFP. </br>
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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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  <a href="http://partsregistry.org/Part:BBa_K1031211">BBa_K1031211</a> is the DmpR transcriptional factor under a constitutive promoter.description  description  description  description  description  description  description  description  description  description  description  description  description  <br/>
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<img src="https://static.igem.org/mediawiki/2015/f/f0/Parts_figure2.jpeg">
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<h6>Figure 1. Metabolic toggle switch to regulate the expression of <em>odhAB</em></h6>
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<p>&nbsp;</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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<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;"><b>Composite part example 2 </b></br>
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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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（consists of basic part3 and basic part4）<a href="http://partsregistry.org/Part:BBa_K1031221">BBa_K1031221</a>, <a href="http://partsregistry.org/Part:BBa_K1031222">BBa_K1031222</a>, <a href="http://partsregistry.org/Part:BBa_K1031223">BBa_K1031223</a> and <a href="http://partsregistry.org/Part:BBa_K1031224">BBa_K1031224</a> are the DmpR biosensors using different RBS preceding sfGFP. </br>
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<img src="https://static.igem.org/mediawiki/2015/2/26/Parts_figure_switch_function_verification.jpeg">
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  <a href="http://partsregistry.org/Part:BBa_K1031211">BBa_K1031211</a> is the DmpR transcriptional factor under a constitutive promoter.description  description  description  description  description  description  description  description  description  description  description  description  description <br/>
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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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                                                 <p>Part-picture-1</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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