Difference between revisions of "Part:BBa K3739032"

Revision as of 23:21, 21 October 2021

J23100-B0030-his-CBM-GFP-B0010

CBM can anchor onto cellulose and GFP can show whether CenA anchor or not.

Usage and Biology

CBM Cellulose enzymes have two domains, and the one that helps bind to cellulose is called cellulose binding module (CBM), and therefore it helps our fusion protein bind to cellulose-rich cell wall. Here we choose the CBM of CenA from Cellulomonas fimi, which has been successfully expressed in Escherichia coli.

GFP Protein tags are peptides genetically fused to the proteins of interest. There are many different kinds of tags developed for different purposes. For labeling experiments, normally the proteins of interest are tagged directly by fluorescent tags. Green fluorescent protein (GFP) make it easier to study protein localization, interactions and dynamics when fusing with other peptides and proteins.

Characterization

In order to eliminate threat from Phaeocystis globosa to the nuclear power plant cooling system, we design a genetic circuit which could express protein aim at this alga. The cellulose binding module (CBM) and (histidine ammonia-lyase) HutH from Pseudomonas Putida were fused to protein CBM-HutH. The fusion protein could bind to the cellulose on the surface of P. globosa and catalyzes L-histidine, secreted by P. globosa to trans-urocanate. which then elevates the ROS around the P. globosa to damage the algal cells.
Promoter (BBa_K525998), RBS (BBa_B0030), his-CBM-HutH gene (BBa_K3739071), and terminator (BBa_B0010) were assembled into pET-28a(+) plasmid backbone to express the his-CBM-HutH. The constructed plasmid was transformed into Vibrio natriegens through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by colony PCR (Fig. 1) and sequencing. Then, the colony with the corrected sequence was cultivated to express HutH target protein. After ultrasonication broke and centrifugation, GE AKTA Prime Plus FPLC System was employed to purify the CBM from the supernatant. Purified protein was verified by electrophoresed on a sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 2). Because CBM own the ability of plastic binding activity, tube and beaker used after ultrasonication are made from glass or Teflon.

Fig. 1. The result of colony PCR. Plasmid pET-28a (+).

Fig. 2. SDS-PAGE analysis of protein his-CBM-HutH. Target bands can be seen at about 69 kDa.

The absorbance of trans-urocanate was measured at 277 nm to obtain the data of concentration. Same concentration and volume of HutH and CBM-HutH solutions were added to the L-histidine solution, and the value OD₂₇₇ was monitored and recorded. As shown in Fig. 3, the OD₂₇₇ in two group was rising continuously, suggesting that CBM-HutH has the ability to catalyzes L-histidine to trans-urocanate.
Fig. 3. Time course of the value of OD₂₇₇.

Sequence and Features