Part:BBa_K4817012:Design
DisH
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 958
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 958
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 958
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 958
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 997
Illegal SapI site found at 1038
Design Notes
The coding sequence of DisH was connected to LacO/LacI (BBa_K1624002, BBa_K3257045) and pT7 (BBa_K4609008). IPTG was used to induce protein expression, simulating quorum sensing-induced protein expression to verify the degradation efficiency of the DisH protein on the membrane. LacO/LacI are commonly found in the pET plasmids. IPTG (isopropyl β-D-1-thiogalactopyranoside) is a molecular analogue of allolactose and cannot be metabolized by common laboratory chassis such as E. coli. IPTG has the same function as allolactose. Both can act as inducers and bind to the repressor in the Lac operon, thereby preventing LacI from binding to LacO upstream of pT7 and ultimately initiating the expression of DisH.
Figure 1. genetic circuit diagram
Figure 2. pET-28a-DisH-C-His plasmid
Source
Desulfovibrio vulgaris
References
[1] Zhu L, Poosarla VG, Song S, Wood TL, Miller DS, Yin B, Wood TK. Glycoside hydrolase DisH from Desulfovibrio vulgaris degrades the N-acetylgalactosamine component of diverse biofilms. Environ Microbiol. 2018 Jun;20(6):2026-2037.
[2] Coffey, B.M., Anderson, G.G. (2014). Biofilm Formation in the 96-Well Microtiter Plate. In: Filloux, A., Ramos, JL. (eds) Pseudomonas Methods and Protocols. Methods in Molecular Biology, vol 1149. Humana, New York, NY.