Difference between revisions of "Team:Nankai/Composite Part"
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<h4>Composite Parts</h4> | <h4>Composite Parts</h4> | ||
<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> | <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> | ||
| − | <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 | + | <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> |
<img src="https://static.igem.org/mediawiki/2015/f/f0/Parts_figure2.jpeg"> | <img src="https://static.igem.org/mediawiki/2015/f/f0/Parts_figure2.jpeg"> | ||
| − | <h6>Figure | + | <h6>Figure 1. Metabolic toggle switch to regulate the expression of <em>odhAB</em></h6> |
| + | <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> | ||
| + | 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> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/2/26/Parts_figure_switch_function_verification.jpeg"> | ||
| + | <h6>Figure 2. Verification of the metabolic toggle switch’s function. IPTG was added to the medium after 12h cultivation.</h6> | ||
Revision as of 14:42, 18 September 2015
Composite Parts
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.
