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sigA strong

sigA strong. sigA. sigA weak. hin. invertase. rcsA. CinR. hixL. hixR. LuxR. LasR. ter---GFP--RBS—rcsA-RBS-SigA-LuxR-hixL- SigA- RBS- Hin-terR-terL-hinR-LasR-SigA-RBS-CinR-RBS-RFP-ter-. (Weak). (Strong). (Activator binding site). (Activator binding site). Other BioBricks.

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sigA strong

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  1. sigA strong sigA sigA weak hin invertase rcsA CinR hixL hixR LuxR LasR ter---GFP--RBS—rcsA-RBS-SigA-LuxR-hixL- SigA- RBS- Hin-terR-terL-hinR-LasR-SigA-RBS-CinR-RBS-RFP-ter- (Weak) (Strong) (Activator binding site) (Activator binding site)

  2. Other BioBricks SigA- RBS- RECDS- ter This codes for the Recombinational Enhancer needed for Hin invertase to work SigA- RBS- Fis CDS- ter This codes for the Fis protein that forms a complex with RE to help Hin Invertase RBS-CinR CDS-ter This codes for the CinR activator protein that will activate our metal container genes SigA- RBS- autoinducer synthase CDS- ter This codes for a protein that makes a quorum sensing molecule which binds in a complex with CinR for Cin Promoter activation CinR Promoter- RBS- FimECDS- GutRCDS- CroCDS- SmtACDS- KinA CDS-ter Construct for metal container decision genes Ter-YFP-Fim-SigG promoter- SigE promoter-Fim-sleBCDS- CwlDCDS-ter Invertible promoter that controls germination gene expression

  3. List of all individual BioBricks: Strong SigA -Lux box 1 Weak SigA -Las box 2 hix –sigA- RBS- hin CDS-ter-ter-hix 3 SigA- RBS- RECDS- ter 4 SigA- RBS- Fis CDS- ter 5 6 RBS-CinR CDS- RBS-RFP-ter SigA- RBS- autoinducer synthase CDS- ter 7 CinR Promoter- RBS- FimECDS- GutRCDS- CroCDS- SmtACDS- KinA CDS-ter 8 Fim-SigG promoter- SigE promoter-Fim-sleBCDS- CwlDCDS-ter 9 10 RBS- rcsACDS-RBS-GFP-ter SigA- RBS- LuxI CDS- ter 11 SigA- RBS- LasI CDS- ter 12

  4. Part sequences • SigA from promoter library- Lux box consensus from Antunes et al.2008 2 . SigA from promoter library- LasR-binding seq • Left hix (Bba_S03383)- sigA- RBS (BBa_K090505)- Hin+LVA (BBa_J31001)- ter-ter-Right Hix (BBa_S03384) • SigA from promoter library- RBS (BBa_K090505)- Recombinational enhancer (BBa_J3101)-ter • SigA from promoter library- RBS (BBa_K090505)- Fis Protein (http://www.ncbi.nlm.nih.gov/nuccore/242375837?from=3271637&to=3271933&report=gbwithparts)-ter • RBS (BBa_K090505)- CinR CDS (BBa_C0077)-ter 7. SigA- RBS (BBa_K090505)- Autoinducer synthase CDS (BBa_C0076)-ter 8. CinR promoter (BBa_R0077)- RBS (BBa_K090505)- Cro CDS (Roberts 1977 paper)-Smta CDS (brick.doc)- kinA CDS (http://www.ncbi.nlm.nih.gov/nuccore/2632216?from=4382&to=6202&report=gbwithparts)- FimE CDS (http://www.uniprot.org/uniprot/P0ADH7.fasta)-ter 9. Fimsite (McCusker et al. 2008)- SigG promoter (DBTBS)-SigE promoter (DBTBS)- SleB CDS http://www.ncbi.nlm.nih.gov/nuccore/1146195?from=12631&to=13548&report=gbwithparts – CwlD CDS http://www.ncbi.nlm.nih.gov/nuccore/1177247?from=567&to=1280&report=gbwithparts. 10 RBS (BBa_K090505)- rcsA CDS (BBa_K137113)- ter 11 SigA- RBS- LuxI (NCBI)-ter 12 SigA- RBS- LasI (NCBI)-ter

  5. Cd_Out: 3000nM Metal Intake Decision:YES

  6. Cd_Out: 3000nM Metal Intake Decision:NO

  7. Lab work: what we hope to prove • Need to show that the sequence flips in the presence of cadmium. We have added GFP and RFP expression to the left and right sides respectively so we can see when the sequence has flipped. • Need to show that there is a biased heads or tails effect happening- GFP should be expressed but a lot less than RFP in the presence of cadmium. • Need to show that the sequence doesn’t flip in the absence of cadmium • Need to show that we can trigger sporulation using the switch (KinA). • Need to show that we can prevent germination in the presence of cadmium (FimE) • Need to show that in the presence of cadmium the FimE invertase flips the promoter for the germination genes and this is why there is no germination (YFP). • Need to show that metallothionein will be located to the spore coat. • Test the switch without using the Cd in the lab. (By adding autoinducers of LuxR and LasR)

  8. Questions • We didn’t use the activator on the left hand side of the switch, as we think it won’t work due to decay of the proteins before they are needed. If they wouldn’t decay when they are in the spore then this would work. • Do we need a link between Spo0A and the metal container decision proteins? • How do we link sensing cadmium to the adjustment of sporulation? • When the metal container decision is ‘No’ we haven’t expressed a protein that will upregulate Cd efflux as we think this will happen in the cell anyway. As an alternative we could express a transcription factor that will repress ArsR and CzrA expression or an activator that can upregulate CadA (ToxR?) • Where to place the RE sequence? • Why are we choosing HixC of Wild type HixL/R (HixC is 16 fold slower than wt) (Davidson BioBrick- BBa_J44000 HixC) • Can we use cadmium in the Lab- otherwise to test our system we need another external control mechanism. Perhaps IPTG-LacI-TetR- CzrA/ArsR orinduce the activators on the left and right hand side of the switch. • Would that be better if we express LuxR and LasR constitutively and express LuxI and LasI upon Cd sensing? This might give us a quicker response.

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