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Team:Dundee/Parts
From 2014.igem.org
BioBricks
Just Another Brick in the Wall
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 pqsA 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
Part:BBa_K1315002 (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 of Xanthomonas 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
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 of Xanthomonas 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.
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
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.
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
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.
Recommended reading:
http://www.ncbi.nlm.nih.gov/pubmed/23285129
http://www.ncbi.nlm.nih.gov/pubmed/20073482
Part:BBa_K1315007 (BCAM0228)
This is the coding sequence of the BCAM0228 two-component regulator of the BDSF sensory BCAM0227-BCAM0228 system. This system is found in Burkholderia cenocepacia. 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.
Recommended reading:
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.
Part:BBa_K1315008 (cblD)
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.
Part:BBa_K1315009
PAI-1 Biosensor 1 (GFP readout)
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 (PAI-1), LasR forms an complex with PAI-1 and it can then activate the PlasB (BBa_R0079) promoter that leads to the production of GFP (BBa_E0040).
Part:BBa_K1315010
PAI-1 Biosensor 2 (GFP readout)
This is a composite part that consists of 4 biobricks. This is an alternative PAI-1 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 (PAI-1), LasR forms an complex with PAI-1 and it can then activate the PluxR (BBa_R0062) promoter that leads to the production of GFP (BBa_E0040).
Welcome to Dundee's 2014 Wiki (Site Information)
Cystic Fibrosis is a genetic disease that results in the accumulation of thick mucus in the epithelial linings of the entire body, particularly the respiratory tract. Over the course of a patient’s life, the mucus-lined lung epithelium becomes repeatedly infected with pathogenic bacteria that stimulate an immune response; leading to inflammation, tissue damage, and ultimately, respiratory failure.
The microflora of the Cystic Fibrosis lung changes over time. In childhood, the major coloniser is Staphylococcus aureus, but as the patient matures other bacterial pathogens infect. The later-dominating pathogens, Pseudomonas aeruginosa and Burkholderia cepacia, are very difficult to eradicate and are associated with chronic decline in lung function. Burkholderia is so infectious that patients have to be isolated from one another, and can be denied lung transplants due to the persistence of this bacterium.
It is important, therefore, for medics to monitor and identify the levels of bacteria within the respiratory tract of a Cystic Fibrosis patient. Currently, patients must provide sputum samples and identification of bacteria takes between 72 hours and 2 weeks, by which point the bacteria can be in an antibiotic resistant state.
The Dundee 2014 iGEM project is focused on designing and testing a device that will rapidly and non-invasively identify the bacteria colonising a Cystic Fibrosis patient. Three biosensors will be developed that recognise external signal molecules produced by key bacteria, all of which are known to be in sputum samples of Cystic Fibrosis patients. A quinolone signal (PQS) is produced by Pseudomonas aeruginosa, a Diffusible Signal Factor (DSF) is produced by Stenotrophomonas maltophilia, an organism that is also associated with Cystic Fibrosis lung infection in adults, and BDSF is a related, but chemically distinct molecule that is produced by Burkholderia cepacia.
By engineering signal recognition and signal transduction proteins from these biological systems the Dundee iGEM team want to produce a portable electronic device that will identify infection outside of the clinic, so that patients do not have to travel great distances, and can be used to help health professionals make informed and rapid decisions on antibiotic treatments.
Read this again by clicking on Site Information in the navigation bar!
Bulletin Board (Policy & Practice)
Come look at the poster explaining how we involved the local and national Cystic Fibrosis community in our project, how we listened to people on the front line of the disease and how they affected our project.
School (Project)
Come in and learn about all the parts that make The Lung Ranger. The lab work that we carried out over the summer is explained here in detail, along with the modelling and implementation.
Library (Log Book)
Read about the experience the team has had over the summer. A week by week description of our work, how we carried out that work and some photos of our time together.
Saloon (iGEM community)
Come on in, take a seat and learn how we have interacted with the larger iGem community. Who the team are, who helped them and how we helped others.
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