Part:BBa_K2759007
AraC/pBAD csgA gs linker hisTag gs linker PBP1b rrnB T1 terminator (Penicillin Binding Peptide)
The penicillin sensitive binding domain of PBP1b is with csgA protein, which is responsible for the formation of amyloid fibers in biofilm structure, and an arabinose inducible promoter.
Usage and Biology
pBAD is an arabinose inducible promoter. Biofilm production by csgA synthesis controlled by presence of arabinose in the environment. csgA has ended with pbp 1b peptide which is expected to interact with the antibiotic when it is present. HisTag is added for western blot analysis.
This composite part is designed for formation of biofilm by bacteria. However, it acquired specialized function: synthesized curli amyloid fibers contains part of the penicillin binding protein domain 1b (PBP 1b). When csgA is expressed, PBP 1b peptide also synthesized at the end of the curli fibers which can interact and hold the antibiotic.
Curli specific gene A(csgA) is the major curli subunit protein that forms amyloid fibers with csgB protein. CsgA can both form homopolymeric and heteropolymeric structure. Homopolymeric structure can go up to several hundered subunit integration. On the other hand, heteropolymeric filaments is generated according to approximate ratio, 20:1 csgA:csgB. CsgB protein is required for heteronucleation.
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.
Curli formation occurs to resist the poor environmental conditions. Therefore, minimal media usage, low temperature and low O2 level can be preferred to trigger curli formation.
Penicillin Binding Protein 1b is major enzyme for peptidoglycan synthesis. Escherichia coli PBP1b is a bifunctional transglycosylase, also known as peptidoglycan glycosyltransferase or murein synthase. It is revealed that particular part of the PBP 1b protein interacts with B-lactam antibiotics.
Characterization
Figure 1: Crystal Violet Assay was done to empty ∆csgA, ∆csgA+original lab part, nc1(new colony 1) and nc2(new colony 2) respectively. All 4 samples were induced with 0%, 4% and 8% arabinose respectively and measurements were done in 3 days and 6 days for all of the replicas
Figure 2: Crystal Violet Assay was done to nc3(new colony 3), nc4(new colony 4), nc5(new colony 5) and cotransformation respectively. All 4 samples were induced with 0%, 4% and 8% arabinose respectively and measurements were done in 3 days and 6 days for all of the replicas
Figure 3: Scanning Electron Microscopy image of empty ΔcsgA cells, uninduced ΔcsgA cells with the plasmid containing the part BBa_K2759007 and induced ΔcsgA cells with the plasmid containing the part BBa_K2759007.
Figure 4: Scanning Electron Microscopy image of ΔcsgA cells with the plasmid containing the part BBa_K2759007. Inductions were done in M63 minimal media supplied with 0.2% L-arabinose. Fibers in the images indicate the formation of biofilm.
Sequence and Features
- 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
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