Difference between revisions of "Part:BBa K2856003"
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To evaluate whether CwaA was involved in adhesion, we performed autoaggregation assays. The cells were collected and washed twice with o.1M PBS (pH=7.0).Then the cells were resuspended in 4ml PBS buffer with an initial OD600=0.5(A0) and the final OD600(At) of the upper most layer was measured after 4 hour cultivation in 30℃ without shaking.<br> | To evaluate whether CwaA was involved in adhesion, we performed autoaggregation assays. The cells were collected and washed twice with o.1M PBS (pH=7.0).Then the cells were resuspended in 4ml PBS buffer with an initial OD600=0.5(A0) and the final OD600(At) of the upper most layer was measured after 4 hour cultivation in 30℃ without shaking.<br> | ||
<ul> | <ul> | ||
| − | + | Autoaggregation (%)=100-(At/A0)X100<br><br> | |
| − | + | ||
[[File:T--H14Z1 Hangzhou--autoaggregation_cwaA.jpeg|400px|thumb|centre| <p>'''Figure. 3 Autoaggregation value of L. lactis NZ9000 and L. lactis/pNZ-cwaA.'''</p>]]<br><br> | [[File:T--H14Z1 Hangzhou--autoaggregation_cwaA.jpeg|400px|thumb|centre| <p>'''Figure. 3 Autoaggregation value of L. lactis NZ9000 and L. lactis/pNZ-cwaA.'''</p>]]<br><br> | ||
Revision as of 12:10, 17 October 2018
Modification of cell wall anchor domain-containing protein (CwaA)
BBa_K2856003 harbors an adhesion-associated protein called cwaA. The cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif.
Usage and Biology
The cwaA gene encodes 923 amino acids with a predicted molecular weight of 93.7 kD.The C terminus of CwaA contains an LPQTDE (LPxTG-like cell wall anchoring) motif belonging to the gram-positive LPxTG anchor superfamily. CwaA possesses five cell wall surface anchor repeat domains. The LPxTG-like motif and three of the five cell wall surface anchor repeat domains. Therefore, CwaA is a cell wall-anchored protein. The specific hit domains of CwaA also included epiglycanin (tandem-repeating region of mucin, pfam05647), OmpC (outer membrane protein, COG3203), PT (the tetrapeptide XPTX repeat, pfam04886) and BF2867_like_N (N-terminal domain found in Bacteroides fragilis Nctc 9343 BF2867 and related proteins, cd13120), probably with a role in cell adhesion
Construction and validation of plasmid pNZ-cwaA
Gene cwaA is from genomic DNA of Lactobacillus plantarum NL42 .In order to remove some illegal restriction enzyme sites and add a HIS tag, the gene cwaA was synthesized by Shanghai Generay Biotech (Shanghai, China). The gene cut with restriction enzyme Hind III and NcoI was ligased with plasmid pNZ8148 cut with the same enzyme. Then the ligation product was transferred to E.coli and spread on plates containing 10 mg/L chloramphenicol. Colonies on the plates were randomly picked and inoculated in 1ml LB medium for 3 hours at 37℃, 200 rpm. 1 μl culture were added to the PCR system as template. As shown in Figure. 2, all the picked colonies had the brand of cwaA, illustrating that the plasmid pNZ-cwaA was successfully constructed.
Validation of autoaggregation of the recombinant L. lactis/pNZ-cwaA
To evaluate whether CwaA was involved in adhesion, we performed autoaggregation assays. The cells were collected and washed twice with o.1M PBS (pH=7.0).Then the cells were resuspended in 4ml PBS buffer with an initial OD600=0.5(A0) and the final OD600(At) of the upper most layer was measured after 4 hour cultivation in 30℃ without shaking.
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Autoaggregation (%)=100-(At/A0)X100
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Sequence and Features