Difference between revisions of "Part:BBa K3799006"
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The plasmid containing R0010 (Plac promoter) was double digested using SpeI and PstI and the plasmid containing I746001 (AIP generator - agrB + agrD with RBSes and terminator) was double restriction digested using Xbal and PstI. The double restriction digestion reaction mixture was incubated for 4hours. | The plasmid containing R0010 (Plac promoter) was double digested using SpeI and PstI and the plasmid containing I746001 (AIP generator - agrB + agrD with RBSes and terminator) was double restriction digested using Xbal and PstI. The double restriction digestion reaction mixture was incubated for 4hours. | ||
| − | [[Image:IISER_Kolkata--digestedplac.jpg | thumb | center | 800 px | ]] | + | [[Image:IISER_Kolkata--digestedplac.jpg | thumb | center | 800 px | Double Digested Plac + plasmid backbone, and double digested agrBD]] |
The digested product was run in 0.8% agarose gel (EtBr) to check the digested products. The digested DNA fragments were purified by agarose gel extraction. | The digested product was run in 0.8% agarose gel (EtBr) to check the digested products. The digested DNA fragments were purified by agarose gel extraction. | ||
The Purified digested products were ligated. The ligated plasmid was transformed into E.Coli DH5a and the cells were plated on an LB agar plate containing Chloramphenicol. Colony PCR was performed to identify the colony containing our desired gene circuit. | The Purified digested products were ligated. The ligated plasmid was transformed into E.Coli DH5a and the cells were plated on an LB agar plate containing Chloramphenicol. Colony PCR was performed to identify the colony containing our desired gene circuit. | ||
| − | [[Image:IISER_Kolkata--plac_agrbd_colonypcr02.jpg | thumb | center | 800 px | ]] | + | [[Image:IISER_Kolkata--plac_agrbd_colonypcr02.jpg | thumb | center | 800 px | Colony PCR of insert(plac+agrBD)]] |
A colony having our desired plasmid was cultured overnight in LB media and the plasmid was extracted by using a miniprep kit. The purified plasmid was transformed into E.Coli BL21 expression vector. Transformed BL21 was cultured in LB media at 37C till 0.6 OD (600 nm) was reached. Protein expression was induced with IPTG. Protein expression was allowed for 10 hours at 30C. The bacterial cells were pellet down and the supernatant containing AIP molecule was filter-sterilised. The supernatant was lyophilised to increase the concentration of the protein content. | A colony having our desired plasmid was cultured overnight in LB media and the plasmid was extracted by using a miniprep kit. The purified plasmid was transformed into E.Coli BL21 expression vector. Transformed BL21 was cultured in LB media at 37C till 0.6 OD (600 nm) was reached. Protein expression was induced with IPTG. Protein expression was allowed for 10 hours at 30C. The bacterial cells were pellet down and the supernatant containing AIP molecule was filter-sterilised. The supernatant was lyophilised to increase the concentration of the protein content. | ||
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===Characterization=== | ===Characterization=== | ||
Revision as of 23:15, 21 October 2021
R0010(plac promoter)+I746001(AIP1 generator - agrB + agrD with RBSes and terminator)
This part consist of the AIP generator(BBa_I746001) under standard IPTG inducible plac promoter(BBa_R0010) of Ecoli.It is used to express S aureus quorum sensing molecule,AIP-1 in Ecoli host. The biosynthesis of AIP requires AgrD, the peptide precursor of AIP, and the integral membrane endopeptidase AgrB.AIP-1 regulates the production of extracellular virulence factors using the agr quorum-sensing system by regulating P2 and P3 promoters.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Quorum sensing in S. aureus is regulated by cyclic thiolactone peptides known as autoinducing peptides (AIPs). The extracellular AIP produced by neighbouring bacteria is sensed by a two-component system encoded in the accessory gene regulator (agr) locus. At high concentration of AIP-1, the cascade is activated, leading to the production of virulence factors including toxins, superantigens, and exo-enzymes.
The biosynthesis of AIP requires AgrD which encodes the peptide precursor of AIP, and an integral membrane endopeptidase AgrB. AgrD propeptide produced initially localizes to the cytoplasmic membrane using the N-terminal amphipathic leader and the AgrB endopeptidase activity removes the AgrD C-terminal tail.
Cloning and Expression
To create this composite part the simple part R0010(Plac promoter) and the I746001 (AIP generator - agrB + agrD with RBSes and terminator) containing pSB1C3 plasmids were obtained from the iGEM DNA Distribution kit. The plasmids were cloned separately in E.Coli DH5α cells and were plated on an LB agar plate containing Chloramphenicol. The primary culture of these transformed bacteria was given in LB media (Chl+) and plasmids were extracted using a miniprep kit. The purified plasmids were stored at -20℃ for future use.
The plasmid containing R0010 (Plac promoter) was double digested using SpeI and PstI and the plasmid containing I746001 (AIP generator - agrB + agrD with RBSes and terminator) was double restriction digested using Xbal and PstI. The double restriction digestion reaction mixture was incubated for 4hours.
The digested product was run in 0.8% agarose gel (EtBr) to check the digested products. The digested DNA fragments were purified by agarose gel extraction. The Purified digested products were ligated. The ligated plasmid was transformed into E.Coli DH5a and the cells were plated on an LB agar plate containing Chloramphenicol. Colony PCR was performed to identify the colony containing our desired gene circuit.
A colony having our desired plasmid was cultured overnight in LB media and the plasmid was extracted by using a miniprep kit. The purified plasmid was transformed into E.Coli BL21 expression vector. Transformed BL21 was cultured in LB media at 37C till 0.6 OD (600 nm) was reached. Protein expression was induced with IPTG. Protein expression was allowed for 10 hours at 30C. The bacterial cells were pellet down and the supernatant containing AIP molecule was filter-sterilised. The supernatant was lyophilised to increase the concentration of the protein content.
