Part:BBa_K1315000 (pqsR)

This is the coding sequence for the transcriptional factor PqsR, which accepts PQS (Pseudomonas quinolone signal). It has been taken from Pseudomonas aeruginosa PAO1. It positively regulates the pqsA promoter (BBa_K1315001), but we did not manage to confirm it.

Usage and Biology
The Pseudomonas quinolone signal (PQS) as well as its precursor 2-heptyl-4-quinolone (HHQ) are very important in mechanisms controlling virulence in Pseudomonas aeruginosa. P. aeruginosa plays a key role in Cystic Fibrosis (CF). PQS can initiate signalling for various processes like virulence, motility, and biofilm formation. A good number of these processes are facilitated by PqsR, which is a transcriptional factor. An activated PqsR will activate the respective promoters, which in our project was pqsA (BBa_K1315001).

Recommended reading:

http://www.ncbi.nlm.nih.gov/pubmed/21539583
http://www.ncbi.nlm.nih.gov/pubmed/?term=pqsr-dependent+and+pqsr-independent+regulation+of+motility+and+biofilm+formation+by+pqs
http://www.ncbi.nlm.nih.gov/pubmed/12393198

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Part:BBa_K1315001 (pqsA)

This is a promoter from Pseudomonas aeruginosa that is positively regulated when the transcription factor PqsR (BBa_K1157001) is activated by PQS (Pseudomonas Quinolone Signal).

Usage and Biology
The Pseudomonas quinolone signal (PQS) is imperative in modulating mechanisms that enable Pseudomonas aeruginosa to survive at times of stress. P. aeruginosa is the main pathogen responsible for severe deterioration in the Cystic Fibrosis lungs. The production of this signal relies on the pqsABCDE operon. The transcriptional factor PqsR (BBa_K1315000) positively regulates this operon.

Recommended Reading:

http://www.ncbi.nlm.nih.gov/pubmed/25225275
http://www.ncbi.nlm.nih.gov/pubmed/16735731
http://www.ncbi.nlm.nih.gov/pubmed/21539583
http://www.ncbi.nlm.nih.gov/pubmed/?term=pqsr-dependent+and+pqsr-independent+regulation+of+motility+and+biofilm+formation+by+pqs
http://www.ncbi.nlm.nih.gov/pubmed/12393198

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Part:BBa_K1315002 (rpfC)

rpfC This is the coding sequence for the RpfC(regulation of pathogenicity factor C) protein. It is the sensor in the two-component signal transduction system ofXanthomonas campestris pv. campestris that activates the regulator RpfG(BBa_K1315003) in the presence of DSF(diffusible signal factor). RpfC eventually leads to the activation of the manA promoter (BBa_K1315006).

Usage and Biology Quorum sensing and bacterial cell signalling are mechanisms known to be utilised by bacteria to regulate their virulence. The RpfC-RpfG sensory system has been found to activate the production of virulence factors like extracellular enzymes and polysaccharides in Xanthomonas campestris. It has been also suggested that this system is involved in linking environmental stimuli, including DSF, with pathogenicity in Xanthomonas campestris. DSF is a cis-unsaturated fatty acid from the DSF family. The RpfC-RpfG two-component system is the most well understood one and is present in all Xanthomonads.

Recommended reading:
http://www.ncbi.nlm.nih.gov/pubmed/?term=r+ryan+dsf+family
http://www.ncbi.nlm.nih.gov/pubmed/?term=A+two-component+system+involving+an+HD-GYP+domain+protein+links+cell%E2%80%93cell+signalling+to+pathogenicity+gene+expression+in+Xanthomonas+campestris

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Part:BBa_K1315003

rpfG This is the coding sequence for the RpfG(regulation of pathogenicity factor G) protein. It is the regulator in the two-component signal transduction system ofXanthomonas campestris pv. campestris that is activated by the sensor kinase RpfC(BBa_K1315002) in the presence of DSF(diffusible signal factor). RpfG eventually leads to the activation of the manA promoter (BBa_K1315006).

Usage and Biology Quorum sensing and bacterial cell signalling are mechanisms known to be utilised by bacteria to regulate their virulence. The RpfC-RpfG sensory system has been found to activate the production of virulence factors like extracellular enzymes and polysaccharides in Xanthomonas campestris. It has been also suggested that this system is involved in linking environmental stimuli, including DSF, with pathogenicity in Xanthomonas campestris. DSF is a cis-unsaturated fatty acid from the DSF family. The RpfC-RpfG two-component system is the most well understood one and is present in all Xanthomonads.

http://www.ncbi.nlm.nih.gov/pubmed/?term=r+ryan+dsf+family
http://www.ncbi.nlm.nih.gov/pubmed/?term=A+two-component+system+involving+an+HD-GYP+domain+protein+links+cell%E2%80%93cell+signalling+to+pathogenicity+gene+expression+in+Xanthomonas+campestris

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Part:BBa_K1315004

ydeH (dgcZ) This is the coding sequence for the zinc-sensory diguanylate cyclase (YdeH). It produces c-di-GMP, which however might prove toxic to E.coli cells when tried to be added. The gene was used to provide some extra cyclic-di-GMP to inhibit Clp(BBa_K1315005).

Usage and Biology Diguanylate cyclases produce cyclic diGMP (c-di-GMP), a secondary messenger. Diguanylate Cyclase (DgcZ), also called YdeH, is present in Escherichia coli and the stimulus that controls it is Zinc.

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Part:BBa_K1315005

clp This is the coding sequence for the cAMP receptor-like protein (Clp). This is a transcriptional activator that is normally inhibited by cyclic di-GMP. Inhibition is alleviated by the RpfG regulator(BBa_K1315003) of the RpfC-RpfG two-component system.

Usage and Biology Quorum sensing and bacterial cell signalling are mechanisms known to be utilised by bacteria to regulate their virulence. The RpfC-RpfG sensory system has been found to activate the production of virulence factors like extracellular enzymes and polysaccharides in Xanthomonas campestris. It has been also suggested that this system is involved in linking environmental stimuli, including DSF, with pathogenicity in Xanthomonas campestris. DSF is a cis-unsaturated fatty acid from the DSF family. The RpfC-RpfG two-component system is the most well understood one and is present in all Xanthomonads. When RpfC(BBa_K1315003) picks up DSF, it eventually signals RpfG(BBa_K1315003) to degrade cyclic di-GMP. This stops the inhibition of Clp and the transcriptional activator can activate genes.

http://www.ncbi.nlm.nih.gov/pubmed/?term=r+ryan+dsf+family
http://www.ncbi.nlm.nih.gov/pubmed/?term=A+two-component+system+involving+an+HD-GYP+domain+protein+links+cell%E2%80%93cell+signalling+to+pathogenicity+gene+expression+in+Xanthomonas+campestris

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Part:BBa_K1315006

manA This is the manA gene promoter taken from Xanthomonas campestris pv. campestris. It is activated by the transcriptional activator Clp (BBa_K1315005).

Usage and Biology The DSF-RpfC(BBa_K1315002)-RpfG(BBa_K1315003) sensory system, in all Xanthomonads, activates the production of the manA gene, which codes for endo-mannanase. It has been shown that the transcriptional regulator Clp(BBa_K1315005), when activated binds to the promoter of manA and turns on its synthesis. Clp is usually inactive by cyclic di-GMP molecules that hinder the ability of the transcriptional factor to bind to DNA.

http://www.ncbi.nlm.nih.gov/pubmed/23285129
http://www.ncbi.nlm.nih.gov/pubmed/20073482

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Part:BBa_K1315007

The BCAM0228 Response Regulator from Burkholderia This is the coding sequence of the BCAM0228 two-component regulator of the BDSF sensory BCAM0227-BCAM0228 system. This system is found in Burkholderiaspp. It is suggested that it activates the cblD promoter(BBa_K1315008).

Usage and Biology The Burkholderia diffusible signal factor (BDSF/cis-2-dodecenoic acid) is a signalling molecule that regulates virulence in Burkholderia cenocepacia. BDSF belongs to the DSF family which was first described in Xanthomonas campestris. BCAM0227 has been identified as a sensor kinase of the BCAM0227-BCAM0228 system that picks up BDSF. However, the confirmed BDSF signalling pathway only goes as far as BCAM0227. It has been proposed that the next steps involve the BCAM0228 regulator protein. The BCAM0228 regulator though is more similar to the OmpR two-component response regulator rather the RpfG(BBa_K1315003) one. Thus, BCAM0228 is the hypothetical regulator of the BCAM0227-BCAM0228 system.

http://www.ncbi.nlm.nih.gov/pubmed/21227698
http://www.ncbi.nlm.nih.gov/pubmed/?term=A+sensor+kinase+recognizing+the+cell-cell+signal+BDSF+(cis-2-dodecenoic+acid)+regulates+virulence+in+Burkholderia+cenocepacia.

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Part:BBa_K1315008

The cblD promoter from Burkholderia This is a promoter that is activated by the OmpR-like two-component response regulator BCAM0228 (BBa_K1315007) in the BCAM0227-BCAM0228 sensor system. The Burkholderia diffusible signal (BDSF) is picked up by the BCAM0227 sensor kinase that is hypothesised to eventually lead in involvement to the phosphorylation of BCAM0228. BCAM0228 then activates transcription from the cblD promoter.

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Part:BBa_K1315009

PA1 Activated System 1 (+GFP) This is a composite part that consists of 4 biobricks. In the front it has ptet(Bba_R0040) which is constitutively on. This produces LasR all the time with lasR(Bba_C0179) next in the sequence. Only in the presence of the Pseudomonas Autoinducer 1 (PA1), LasR forms an complex with PA1 and it can then activate the plasB(Bba_R0079) promoter that leads to the production of GFP(Bba_E0040).

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Part:BBa_K1315010

PA1 Activated System 2 (+GFP)
This is a composite part that consists of 4 biobricks. This is an alternative PA1 Activated System from Bba_K1315009 that instead uses pluxR (Bba_R0062). In the front it has ptet(Bba_R0040) which is constitutively on. This produces LasR all the time with lasR(Bba_C0179) next in the sequence. Only in the presence of the Pseudomonas Autoinducer 1 (PA1), LasR forms an complex with PA1 and it can then activate the pluxR(Bba_R0062) promoter that leads to the production of GFP(Bba_E0040).