sigA strong

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sigA strong
sigA
sigA weak
hin
invertase
rcsA
CinR
hixL
hixR
LuxR
LasR
ter---GFP--RBSrcsA-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)
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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
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List of all individual BioBricks
Strong SigA -Lux box
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Weak SigA -Las box
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hix sigA- RBS- hin CDS-ter-ter-hix
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SigA- RBS- RECDS- ter
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SigA- RBS- Fis CDS- ter
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RBS-CinR CDS- RBS-RFP-ter
SigA- RBS- autoinducer synthase CDS- ter
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CinR Promoter- RBS- FimECDS- GutRCDS- CroCDS-
SmtACDS- KinA CDS-ter
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Fim-SigG promoter- SigE promoter-Fim-sleBCDS-
CwlDCDS-ter
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RBS- rcsACDS-RBS-GFP-ter
SigA- RBS- LuxI CDS- ter
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SigA- RBS- LasI CDS- ter
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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)-
  HinLVA (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/2
  42375837?from3271637to3271933reportgbwithpart
  s)-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?from4382to6202reportgbwithpa
  rts)- FimE CDS (http//www.uniprot.org/uniprot/P0A
  DH7.fasta)-ter
• 9. Fimsite (McCusker et al. 2008)- SigG promoter
  (DBTBS)-SigE promoter (DBTBS)- SleB CDS
  http//www.ncbi.nlm.nih.gov/nuccore/1146195?from1
  2631to13548reportgbwithparts CwlD CDS
  http//www.ncbi.nlm.nih.gov/nuccore/1177247?from5
  67to1280reportgbwithparts.
• 10 RBS (BBa_K090505)- rcsA CDS (BBa_K137113)- ter
• 11 SigA- RBS- LuxI (NCBI)-ter

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Cd_Out 3000nM Metal Intake DecisionYES
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Cd_Out 3000nM Metal Intake DecisionNO
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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 doesnt 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)

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Questions

• We didnt use the activator on the left hand side
  of the switch, as we think it wont work due to
  decay of the proteins before they are needed. If
  they wouldnt 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
  havent 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.