Difference between revisions of "Part:BBa K1628002"

Latest revision as of 07:49, 18 September 2015

Pxyl

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

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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K1628002 short</partinfo>
-Promoter Pxyl is a promoter of xylose operon regulate by repressor XylR. Promoter Pxyl together with promoter PlacI, repressor LacI (BBa_K1628201), promoter Pgrac (BBa_K1628202) and repressor XylR (BBa_K1628203) formed a metabolic toggle switch. We used this device to regulate the expression of odhAB genes in B. amyloliquefaciens NK-1 (showed in Figure 2).
+Promoter Pxyl is a promoter of xylose operon regulate by repressor XylR. Promoter Pxyl together with promoter PlacI, repressor LacI (BBa_K1628201), promoter Pgrac (BBa_K1628202) and repressor XylR (BBa_K1628203) formed a metabolic toggle switch. We used this device to regulate the expression of ''odhAB'' genes in ''B. amyloliquefaciens'' NK-1 (showed in Figure 1).
+[[File:switch1_NK.png]]
+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.
+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.
+[[File:switch2_NK.png]]
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