Transcript Slide 1

sigA strong
sigA
sigA weak
hin invertase
rcsA
hixL
LuxR
CinR
hixR
LasR
ter---GFP--RBS—rcsA-RBS-SigA-LuxR-hixL- SigA- RBS- Hin-terR-terL-hinR-LasR-SigA-RBS-CinR-RBS-RFP-ter(Strong)
(Activator binding site)
(Weak)
(Activator binding site)
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
List of all individual BioBricks:
1
2
Strong SigA -Lux box
Weak SigA -Las box
hix –sigA- RBS- hin CDS-ter-ter-hix
3
4
5
6
7
SigA- RBS- RECDS- ter
SigA- RBS- Fis CDS- ter
RBS-CinR CDS- RBS-RFP-ter
SigA- RBS- autoinducer synthase CDS- ter
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
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
RBS (BBa_K090505)- CinR CDS (BBa_C0077)-ter
6.
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
4000
mRNA A rsR
ArsR
mRNA CzrA
CinR
3500
CzrA
mRNA CI
MntH
CI
3000
CI
mRNA LuxRToxR
Spo0A
Cd
LuxR
mRNA Hin
Hin
ArsRCd
2500
CzrACd
LasR
mRNA HinToLeft
2000
mRNA RcsA
RcsA
mRNA CinR
1500
CinR
TetR
mRNA KinA
KinA
mRNA Spo0A
1000
Cro
Spo0A
mRNA SmtACroFimEGutR
ArsR
CadA
500
SmtA
Cro
FimE
GutR
mRNA MntH
Hin
LuxR
0
0
1000
2000
3000
4000
5000
MntH
mRNA CadA
CadA
CdInside
Cd_Out:
3000nM
Metal Intake
Decision:YES
25
mRNA A rsR
ArsR
mRNA CzrA
CzrA
mRNA CI
CI
mRNA LuxRToxR
20
LuxR
mRNA Hin
Hin
ArsRCd
mRNA-CinR
15
CzrACd
LasR
mRNA HinToLeft
mRNA RcsA
RcsA
mRNA CinR
10
CinR
TetR
mRNA KinA
KinA
mRNA Spo0A
Spo0A
mRNA SmtACroFimEGutR
5
SmtA
Cro
FimE
GutR
mRNA MntH
mRNA-RcsA
0
0
1000
2000
3000
4000
5000
MntH
mRNA CadA
CadA
CdInside
450
mRNA A rsR
ArsR
mRNA CzrA
400
CzrA
mRNA CI
CI
mRNA LuxRToxR
350
LuxR
mRNA Hin
300
Hin
ArsRCd
CzrACd
250
LasR
mRNA HinToLeft
mRNA RcsA
Hin
200
RcsA
mRNA CinR
CinR
TetR
mRNA KinA
150
KinA
mRNA Spo0A
100
Spo0A
mRNA SmtACroFimEGutR
50
SmtA
Cro
FimE
GutR
mRNA MntH
mRNA-CinR
mRNA-RcsA
0
0
1000
2000
3000
4000
5000
MntH
mRNA CadA
CadA
CdInside
4000
mRNA A rsR
MntH
ArsR
mRNA CzrA
3500
CzrA
mRNA CI
CI
mRNA LuxRToxR
3000
LuxR
mRNA Hin
Hin
ArsRCd
2500
CzrACd
LasR
mRNA HinToLeft
2000
mRNA RcsA
RcsA
mRNA CinR
1500
CinR
TetR
mRNA KinA
CadA
KinA
mRNA Spo0A
Cd
1000
Spo0A
mRNA SmtACroFimEGutR
ArsR
SmtA
Cro
FimE
GutR
mRNA MntH
500
Hin
LuxR
0
0
1000
2000
3000
4000
5000
MntH
mRNA CadA
CadA
CdInside
Cd_Out:
3000nM
Metal Intake
Decision:NO
14
mRNA A rsR
ArsR
mRNA CzrA
CzrA
mRNA CI
12
CI
mRNA LuxRToxR
LuxR
mRNA Hin
10
Hin
ArsRCd
CzrACd
8
LasR
mRNA HinToLeft
mRNA RcsA
RcsA
mRNA CinR
6
CinR
TetR
mRNA KinA
mRNA-RcsA
4
KinA
mRNA Spo0A
Spo0A
mRNA SmtACroFimEGutR
2
SmtA
Cro
FimE
GutR
mRNA MntH
mRNA-CinR
0
0
1000
2000
3000
4000
5000
MntH
mRNA CadA
CadA
CdInside
450
mRNA A rsR
ArsR
mRNA CzrA
400
CzrA
mRNA CI
CI
mRNA LuxRToxR
350
LuxR
mRNA Hin
Hin
300
Hin
ArsRCd
CzrACd
250
LasR
mRNA HinToLeft
mRNA RcsA
200
RcsA
mRNA CinR
CinR
TetR
mRNA KinA
150
KinA
mRNA Spo0A
100
Spo0A
mRNA SmtACroFimEGutR
SmtA
Cro
FimE
GutR
mRNA MntH
50
mRNA-RcsA
0
0
1000
2000
mRNA-CinR
3000
4000
5000
MntH
mRNA CadA
CadA
CdInside
25
mRNAA rsR
ArsR
mRNA CzrA
CzrA
mRNACI
mRNA-CinR
20
CI
mRNA LuxRToxR
LuxR
mRNAHin
Hin
ArsRCd
15
CzrACd
LasR
mRNAHinToLeft
mRNA-RcsA
mRNARcsA
RcsA
mRNACinR
10
CinR
TetR
mRNAKinA
KinA
mRNASpo0A
Spo0A
mRNASmtA CroFimEGutR
5
SmtA
Cro
FimE
GutR
mRNAMntH
0
0
500
1000
1500
2000
2500
3000
MntH
3500 mRNA
4000adA
C
CadA
CdInside
4500
5000
4000
mRNAA rsR
3500
ArsR
mRNACzrA
mnTH
CzrA
mRNACI
3000
Spo0A
CI
mRNA LuxRToxR
LuxR
mRNAHin
2500
Cd
Hin
ArsRCd
2000
CzrACd
LasR
mRNAHinToLeft
1500
mRNARcsA
CinR
1000
ArsR
RcsA
mRNACinR
CadA
500
CinR
TetR
mRNAKinA
Hin
LuxR
0
0
1000
2000
3000
4000
5000
KinA
mRNASpo0A
Spo0A
mRNASmtACroFimEGutR
SmtA
Cro
FimE
GutR
mRNAMntH
MntH
mRNA adA
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)
Questions
1. 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.