Upload
luna
View
53
Download
2
Embed Size (px)
DESCRIPTION
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. - PowerPoint PPT Presentation
Citation preview
sigA strong
hixL
sigA weak
hixR
hin invertase
CinR
sigA
LuxR LasR
rcsA
ter---GFP--RBS—rcsA-RBS-SigA-LuxR-hixL- SigA- RBS- Hin-terR-terL-hinR-LasR-SigA-RBS-CinR-RBS-RFP-ter-
(Strong) (Weak)
(Activator binding site) (Activator binding site)
RBS-CinR CDS-terThis 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
SigA- RBS- RECDS- terThis codes for the Recombinational Enhancer needed for Hin invertase to work
SigA- RBS- Fis CDS- terThis codes for the Fis protein that forms a complex with RE to help Hin Invertase
CinR Promoter- RBS- FimECDS- GutRCDS- CroCDS- SmtACDS- KinA CDS-terConstruct for metal container decision genes
Ter-YFP-Fim-SigG promoter- SigE promoter-Fim-sleBCDS- CwlDCDS-terInvertible promoter that controls germination gene expression
Other BioBricks
Strong SigA -Lux box
hix –sigA- RBS- hin CDS-ter-ter-hix
Weak SigA -Las box
1
2
3
6
9
RBS-CinR CDS- RBS-RFP-ter
Fim-SigG promoter- SigE promoter-Fim-sleBCDS- CwlDCDS-ter
8 CinR Promoter- RBS- FimECDS- GutRCDS- CroCDS- SmtACDS- KinA CDS-ter
4 SigA- RBS- RECDS- ter
5 SigA- RBS- Fis CDS- ter
7 SigA- RBS- autoinducer synthase CDS- ter
10 RBS- rcsACDS-RBS-GFP-ter
List of all individual BioBricks:
11 SigA- RBS- LuxI CDS- ter
12 SigA- RBS- LasI CDS- ter
Part sequences 1. SigA from promoter library- Lux box consensus from Antunes et al.2008
2 . SigA from promoter library- LasR-binding seq
3. Left hix (Bba_S03383)- sigA- RBS (BBa_K090505)- Hin+LVA (BBa_J31001)- ter-ter-Right Hix (BBa_S03384)
4. SigA from promoter library- RBS (BBa_K090505)- Recombinational enhancer (BBa_J3101)-ter
5. SigA from promoter library- RBS (BBa_K090505)- Fis Protein (http://www.ncbi.nlm.nih.gov/nuccore/242375837?from=3271637&to=3271933&report=gbwithparts)-ter
6. 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)- ter11 SigA- RBS- LuxI (NCBI)-ter12 SigA- RBS- LasI (NCBI)-ter
Cd_Out:3000nM
Metal IntakeDecision:YES
0 1000 2000 3000 4000 50000
500
1000
1500
2000
2500
3000
3500
4000
mRNA
ArsR
ArsR
mRNACzrA
CzrA
mRNACI
CI
mRNA LuxR
ToxR
LuxRmRNA
Hin
Hin
ArsRCd
CzrACd
LasR
mRNAHin
To
Left
mRNARcsA
RcsA
mRNACinR
CinRTetR
mRNAKinA
KinA
mRNASpo0A
Spo0A
mRNASmtA
Cro
FimE
GutR
SmtACro
FimE
GutRmRNA
MntH
MntH
mRNACadA
CadACd
Inside
MntH
CinR
Cd
Cro
CadA
ArsR
LuxR
Hin
Spo0A
CI
0 1000 2000 3000 4000 50000
5
10
15
20
25
mRNAArsR
ArsR
mRNACzrA
CzrA
mRNACI
CI
mRNA LuxR
ToxR
LuxRmRNAHin
Hin
ArsRCd
CzrACd
LasR
mRNAHin
To
Left
mRNARcsA
RcsA
mRNACinR
CinRTetR
mRNAKinA
KinA
mRNASpo0A
Spo0A
mRNASmtA
Cro
FimE
GutR
SmtACro
FimE
GutRmRNA
MntH
MntH
mRNACadA
CadACd
Inside
mRNA-RcsA
mRNA-CinR
0 1000 2000 3000 4000 50000
50
100
150
200
250
300
350
400
450
mRNAArsR
ArsR
mRNACzrA
CzrA
mRNACI
CI
mRNA LuxR
ToxR
LuxRmRNAHin
Hin
ArsRCd
CzrACd
LasR
mRNAHin
To
Left
mRNARcsA
RcsA
mRNACinR
CinRTetR
mRNAKinA
KinA
mRNASpo0A
Spo0A
mRNASmtA
Cro
FimE
GutR
SmtACro
FimE
GutRmRNA
MntH
MntH
mRNACadA
CadACd
Inside
mRNA-RcsAmRNA-CinR
Hin
0 1000 2000 3000 4000 50000
500
1000
1500
2000
2500
3000
3500
4000
mRNAArsR
ArsR
mRNACzrA
CzrA
mRNACI
CI
mRNA LuxRToxR
LuxRmRNAHin
Hin
ArsRCd
CzrACd
LasR
mRNAHin
To
Left
mRNARcsA
RcsA
mRNACinR
CinRTetR
mRNAKinA
KinA
mRNASpo0A
Spo0A
mRNASmtACroFimEGutR
SmtACro
FimE
GutRmRNA
MntH
MntH
mRNACadA
CadACd
Inside
MntH
CadA
Cd
ArsR
LuxRHin
Cd_Out:3000nM
Metal IntakeDecision:NO
0 1000 2000 3000 4000 50000
2
4
6
8
10
12
14
mRNA
ArsR
ArsR
mRNACzrA
CzrA
mRNACI
CI
mRNA LuxR
ToxR
LuxRmRNA
Hin
Hin
ArsRCd
CzrACd
LasR
mRNAHin
To
Left
mRNARcsA
RcsA
mRNACinR
CinRTetR
mRNAKinA
KinA
mRNASpo0A
Spo0A
mRNASmtA
Cro
FimE
GutR
SmtACro
FimE
GutRmRNA
MntH
MntH
mRNACadA
CadACd
Inside
mRNA-CinR
mRNA-RcsA
0 1000 2000 3000 4000 50000
50
100
150
200
250
300
350
400
450
mRNA
ArsR
ArsR
mRNACzrA
CzrA
mRNACI
CI
mRNA LuxR
ToxR
LuxRmRNA
Hin
Hin
ArsRCd
CzrACd
LasR
mRNAHin
To
Left
mRNARcsA
RcsA
mRNACinR
CinRTetR
mRNAKinA
KinA
mRNASpo0A
Spo0A
mRNASmtA
Cro
FimE
GutR
SmtACro
FimE
GutRmRNA
MntH
MntH
mRNACadA
CadACd
Inside
mRNA-CinR
mRNA-RcsA
Hin
0 500 1000 1500 2000 2500 3000 3500 4000 4500 50000
5
10
15
20
25
mRNA
ArsR
ArsR
mRNACzrA
CzrAmRNA
CI
CI
mRNA LuxR
ToxR
LuxR
mRNAHin
HinArsR
Cd
CzrACd
LasRmRNA
Hin
To
Left
mRNARcsA
RcsA
mRNACinR
CinR
TetR
mRNAKinA
KinA
mRNASpo0A
Spo0AmRNA
SmtA
Cro
FimE
GutR
SmtA
CroFimE
GutR
mRNAM
ntH
MntH
mRNACadA
CadACd
Inside
mRNA-CinR
mRNA-RcsA
0 1000 2000 3000 4000 50000
500
1000
1500
2000
2500
3000
3500
4000
mRNA
ArsR
ArsR
mRNACzrA
CzrA
mRNACI
CI
mRNA LuxR
ToxR
LuxRmRNA
Hin
Hin
ArsRCd
CzrACd
LasR
mRNAHinToLeft
mRNARcsA
RcsA
mRNACinR
CinRTetR
mRNAKinA
KinA
mRNASpo0A
Spo0A
mRNASmtA
Cro
FimE
GutR
SmtACro
FimE
GutRmRNA
MntH
MntH
mRNACadA
CadACdInside
mnTH
Spo0A
Cd
CadA
CinR
ArsR
LuxR
Hin
Lab work: what we hope to prove
1. 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.
2. 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.
3. Need to show that the sequence doesn’t flip in the absence of cadmium
4. Need to show that we can trigger sporulation using the switch (KinA).
5. Need to show that we can prevent germination in the presence of cadmium (FimE)
6. 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).
7. Need to show that metallothionein will be located to the spore coat.
8. Test the switch without using the Cd in the lab. (By adding autoinducers of LuxR and LasR)
Questions1. 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.
2. Do we need a link between Spo0A and the metal container decision proteins?
3. How do we link sensing cadmium to the adjustment of sporulation?
4. 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?)
5. Where to place the RE sequence?
6. Why are we choosing HixC of Wild type HixL/R (HixC is 16 fold slower than wt) (Davidson BioBrick- BBa_J44000 HixC)
7. 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.
8. 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.