Part:BBa_K2759007:Design
AraC/pBAD csgA gs linker hisTag gs linker PBP1b rrnB T1 terminator (Penicillin Binding Peptide)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
Illegal XhoI site found at 1782 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
Design Notes
Site directed mutagenesis is performed on csgA to eliminate EcoRI cutsite.
csgA-pbp1b is added to an inducible promoter rather than a constitutive one in order to prevent any toxicity risk that could be posed on cells by excess biofilm formation.
Use competent cell that has csgA deletion to make functional this gene.
Curli is resistant to proteolytic cleavage or dissolution by sodium dodecyl sulphate(SDS). Therefore SDS-PAGE cannot give accurate results to see curli amloid protein formation. It requires harsh treatment with formic acid or hexafluoroisopropanol to depolymerize the fibers to obtain subunits.
Curliated bacteria can be stained red by congo red dye. Therefore, biofilm production can be detected by congo red staining. Alternatively, scanning electron microscope(SEM) might be used to visuaize the fibers in the extracellular area.
Penicillin Binding Peptide is comprise of 8 amino acids with stop codon. Since the length of the PBP is short, we add HisTag by using GS linker in order not to disrupt the 3D configuration of the PBP.
Source
Escherichia coli (strain K12)- Nicholas et al. Purification and Sequencing of the Active Site Tryptic Peptide from Penicillin-Binding Protein 1 b of Escherichia coli. 1985 csgA gene is obtained E. Coli K-12.
References
Barnhart, Michelle M., and Matthew R. Chapman. "Curli biogenesis and function." Annu. Rev. Microbiol. 60 (2006): 131-147.
Van Gerven, Nani, et al. "Bacterial amyloid formation: structural insights into curli biogensis." Trends in microbiology 23.11 (2015): 693-706. Nicholas, Robert A., et al. "Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli." Biochemistry 24.14 (1985): 3448-3453.
Charpentier, Xavier, et al. "Penicillin-binding proteins 1a and 1b form independent dimers in Escherichia coli." Journal of bacteriology 184.13 (2002): 3749-3752.
Sung, Ming-Ta, et al. "Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli." Proceedings of the National Academy of Sciences 106.22 (2009): 8824-8829.