Difference between revisions of "Part:BBa K118022:Experience"

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Applications of BBa_K118022

Characterization

Edinburgh 2011 conducted two assays, comparing the activity of this part (but under the control of the lac promoter, giving BBa_K523016) to a β-glucosidase (E. coli bglX, also under the control of the lac promoter) on two different substrates:

4-methylumbelliferyl β- D- glucuronide (MUG, left photo). This substrate is a cellobiose analog.
4-methylumbelliferyl β- D- cellobioside (MUC, right photo). This substrate is larger and is more like a cellulose analog.

Both substrates produce a fluorescent product when cleaved. Our plates below show the results of placing cell lysate and cell debris on an MUG plate and an MUC plate. Present on both plates are:

Left side of plate: lysate/debris from JM109 expressing bglX, BBa_K523014
Right side of plate: lysate/debris from JM109 expressing cex, BBa_K523016
Bottom of plate: lysate/debris from JM109 cells


MUG assay. bglX on left, cex on right.		MUC assay. bglX on left, cex on right.

As can be seen, cex is much better at degrading MUC.

Further Characterization of BBa_K118022 on S-layer of Caulobacter crescentus

Contributed by British_Columbia 2016 iGEM team.

We have selected CEX protein to be displayed on the surface of Caulobacter crescentus for transformation of lignocellulose into fermentable sugars. The CEX construct from 43 to 443 amino acids position was cloned into rsaA protein in p4A723 plasmid and transformed into Caulobacter crescentus. The cellulase activity was tested in a test with 2,4-dinitrophenylcellobiose(DNPC) substrate, which consists of cellubiose bound to a chromophore. If cellulase activity is present, the release of chromophore can be observed and measured at OD400. CEX displayed on the C.crescentus surface showed significantly higher activity than wild type rsaA. The results of the assay after 12 hours are shown on the Picture.

Characterization from iGEM19_CAU_China

We linke the cex gene BBa_K118022 into a pET30a(+) backbone, then transforme this plasmid into BL21. After the overnight (about 15h) induction under the condition of 16℃ 0.08 mM, we smash the recombinant E.coli strain with the ultrasonication to get crude enzyme solution, then we measured the enzyme activity by the method of CMC-Na(sodium carboxymethyl cellulose) assey. The result is shown on Fig.3[1].

User Reviews

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+===Characterization from iGEM19_CAU_China===
+We linke the cex gene BBa_K118022 into a pET30a(+) backbone, then transforme this plasmid into BL21. After the overnight (about 15h) induction under the condition of 16℃ 0.08 mM, we smash the recombinant E.coli strain with the ultrasonication to get crude enzyme solution, then we measured the enzyme activity by the method of CMC-Na(sodium carboxymethyl cellulose) assey. The result is shown on Fig.3[1].
 ===User Reviews===