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Team:Groningen/Template/MODULE/project/parts
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Parts
Detection system
We want Lactococcus lactis to start producing growth inhibiting molecules only when a pathogen is present. Therefore, L. lactis must be able to sense Pseudomonas aeruginosa and Staphylococcus aureus.
Detecting Pseudomonas aeruginosa
To give Lactococcus lactis the ability to detect P. aeruginosa we assembled a new BioBrick. We adopted a widely used principle for detection, namely detection of quorum molecules. P. aeruginosa produces AHLs for communication1, therefore we have built an AHL sensor) that can be used by L. lactis.
To test the how well Lactococcus lactis can detect P. aeruginosa we build a construct which has the AHL sensor ability coupled with a GFP gene.
A related part that was made is the AHL generator. Many iGEM teams are working with AHL sensing, either to detect P. aeruginosa or for an regulatory mechanisms. Our AHL generator produces AHLs, which enables other teams to produce AHLs and test their sensors without the hassle of working with the pathogenic bacterium itself.
Lactococcus lactis toolbox
Basic BioBricks were submitted that form the basis for working with Lactococcus lactis as a chassis. For getting BioBricks in L. lactis a vector, pIL253 with mRFP was made. Another completely new BioBrick is the sfGFP(Bs) that is highly efficient in L. lactis. Also, some genes of the nisin operon, NisA, NisC, NisR and NisK were BioBricked.
Secretion system
Infection by Staphylococcus aureus and Pseudomonas aeruginosa will be battled by Lactococcus lactis using molecules that prevent biofilm formation, disrupt communication between pathogens and kill the pathogen.
Antibiofilm formation
When bacteria start infection of a wound, they often protect themselves with a biofilm. For an effective fight against these infections, the biofilm should be broken down. We have assembled a BioBrick of dispersinB fused to an USP45tag. This part was later assembled in the secretion system, and can work against S. aureus and P. aeruginosa .
Battling Pseudomonas aeruginosa
We will fight against P. aeruginosa with an dual action system, consisting of the new BioBricks dispersinB and aiiA. DispersinB works against biofilm formation as described above. AiiA is an quorum quenching enzyme which disrupt communication between P. aeruginosa. By disrupting communication we try reduce the activity of both dividing and activation of pathogenic genes by P. aeruginosa.2
Battling Staphylococcus aureus
The cell wall of the Gram-positive bacterium S. aureus contains Lipid-2 fatty acids. These are perfect targets for the anti-microbial nisin.3 We designed and assembled nisin in an a novel BioBrick that combines the expression of dispersinB and nisA. With this second dual action system, we want to fight of infections by S. aureus.
More about
References
1. Toyofuku, M., Uchiyama, H. and Nomura, N. (2012) Social Behaviours under Anaerobic Conditions in
Pseudomonas aeruginosa. Int. J. Microbiol. 2012: 405191
2. Wang, Y. et al. (2007) Effects of quorum sensing autoinducer degradation gene on
virulence and biofilm formation of Pseudomonas aeruginosa. Sci. China C. Life Sci. 50: 385-391
3. Wiedemann, I., Benz, R. and Sahl, H.G. (2004) Lipid II-Mediated Pore Formation by the Peptide Antibiotic Nisin:
a Black Lipid Membrane Study. J. Bacteriol. 186: 3259-3261
4. Overkamp, W. et al. (2013) Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging. Appl. Environ. Microbiol. 79: 6481-6490
5. Borrero, J. et al. (2011) Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis. Appl. Microbiol. Biotechnol. 89: 131-143
6. Simon, D. and Chopin, A. (1988) Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie 70: 559-566
Submitted parts
| Part | Type | Description | Designer | Length | |
|---|---|---|---|---|---|
| BBa_K1365000 | Coding | nisA | Nisin precursor. Should be used in combination with the other genes on the nisin operon (e.g. in Lactococcus lactis NZ9800). | Sandra Mous | 174 |
| BBa_K1365003 | Coding | nisC | Protein needed in the cyclization of the nisin precursor. Part of the nisin operon. | Sandra Mous | 1245 |
| BBa_K1365006 | Coding | nisRK | Two proteins involved in the recognition of nisin and positive feedback regulation to induce the PNisA promoter. This BioBrick is part of the nisin operon. | Sandra Mous | 2023 |
| BBa_K1365020 | Coding | sfGFP(Bs) | SfGFP(Bs) is a for Bacillus optimized version of the high performance sfGFP that works really well in Lactococcus lactis.4 | Sandra Mous | 1625 |
| BBa_K1365100 | Device | P2-nisA-dspB-ssUsp45 | Dual action system against S. aureus, made for expression in L. lactis. Codes for the nisin precursor NisA and the biofilm degrading enzyme DspB. The latter is fused with an USP45sp for transport out of the cell. This device has an P2 promoter and is thus transcribed after binding with p-AgrA. | Rick Elbert | 1625 |
| BBa_K1365111 | Coding | ssUsp45-dspB | DispersinB fused to an Usp45sp. The DspB enzyme is secreted from the cell via the USP system of L. lactis.5 | Rick Elbert | 1170 |
| BBa_K1365120 | Device | PLasR-aiiA-dpsB | Dual action system against P. aeruginosa for expression in L. lactis and includes the genes for the autoinducer inactivation enzyme A (AiiA) and Dispersin B (DspB). | Rick Elbert | 2350 |
| BBa_K1365130 | Device | PLasR-aiiA-dspB-his | Same system as BBa_K1365120, but with 6x histidine tags fused to the two proteins. | Rick Elbert | 2416 |
| BBa_K1365169 | Coding | ssUsp45-aiiA | Autoinducer inactivation enzyme A (AiiA) gene fused to signalsequence for the USP45 secretion pathway. | Rick Elbert | 834 |
| BBa_K1365301 | Plasmid | pIL253 | Shuttlevector for L. lactis and B. subtilis.6 Includes the BioBrick compatible VF2 and RV primer binding sites, prefix and suffix. Contains no illegal restriction sites. | Lisa Hielkema | 6230 |
| BBa_K1365555 | Composition | RBS-sfGFP-dt | SfGFP(Bs) combined with RBS and double terminator. This part was used to assemble sfGFP(Bs) with different promoters to characterize the promoters. | Aakanksha Saraf | 874 |
| BBa_K1365556 | Composition | RBS-nisA-dt | NisA combined with an RBS and double terminator. This part was assembled with different promotors for characterization experiments. | Aakanksha Saraf | 328 |
| BBa_K1365557 | Reporter | J23101-sfgfp(bs) | SfGFP(Bs) with the constitutive promoter J23101. Designed for use in L. lactis and E. coli. | Aakanksha Saraf | 915 |
| BBa_K1365997 | Device | lasR | AHL sensor. The LasR membrane protein is produced by this device. | Chandhuru Jagadeesan | 1003 |
| BBa_K1365998 | Signaling | lasI | AHL generator. This part can be used to mimic the presence of the AHL producing bacteria P. aeruginosa. | Chandhuru Jagadeesan | 892 |
| BBa_K1365999 | Composition | RBS-GFP-dt | GFP with RBS and double terminator. This BioBrick is compatible with L. lactis and E. coli. | Chandhuru Jagadeesan | 873 |
