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.