Plasmids - Genetic circuits built in this study

2.6 Genetic circuits built in this study

2.6.2 Plasmids

For E. coli and B. subtilis studies, we used or built the plasmids listed in Table 2.1. Plasmids were always propagated in E. coli DH5α at 37◦_{C unless stated otherwise in}

the plasmid notes (for instance, temperature sensitive replicons). The R6k origin of replication plasmids were propagated in an E. coli DH5α λpir strain to allow them to replicate[133]. Plasmids were usually constructed in DH5α and characterised in E. coli MG1655 or BW25113 strains[12].

Chapter 2. Backgr ound and genetic library engineering

Source Label Template Modification Marker Note

[195] pACYC184 Cam, Tet Cloning template

[253] pUC19 Amp Cloning template

[238] pBR322 Amp Cloning template

[187] pSB1A2 Amp Cloning template

[187] pSB1C3 Cloning

template

[187] pSB1AK3 Amp, Kan mCherry LuxR with LVA degradation

tail

[90] pBad33 Cam Cloning template for low-copy num-

ber circuits with inducible Pbad sys- tem

[43] pKD46 Amp thermosensitive plasmid with 30deg

growth and propagation

Demuris pIJ790 Cam thermosensitive plasmid with 30deg

growth and propagation

[32] pCP20 Amp Frt-site recombinase, temperature

sensitive

This study pCWqs01 pACYC184 B0015 Cam Cloning template for low-copy num-

ber circuits

This study pCWqs02 pSB1AK3-B0015 B0015 x3 Amp Cloning template for high-copy num- ber circuits

2.6. Genetic cir cuits built in this study 33

This study pCWqs03 pACYC184-B0015 Cam sender-CI without TetR constitutive

expression This study pCWqs04 pSB1AK3-B0015x2 Prlux-B0034-

mCherry

Amp mCherry under control of Prlux This study pCWqs05 pSB1AK3-B0015x2 J23119-B0034-

LuxR

Amp LuxR under the control of strong constitutive promoter

This study pCWqs06 pSB1AK3-B0015x2 J23119-B0034- LuxR-Prlux- B0034-mCherry

Amp, Kan mCherry LuxR with LVA degradation tail

This study pCWqs07 pSB1C3 PltetO1-B0034-

LuxI

Cam LuxI with LVA degradation tail

This study pCWqs08 pSB1C3 B0034-GFPlva-

B0015

Cam GFP with lva degradation tail This study pCWqs09 pACYCB15 J23119-B34-TetR Cam TetR with LVA degradation tail This study pCWqs10 pSB1C3 Plteto1-B34-LuxI-

B34-GFP-B15

Cam inducible signal of C6-HSL couple with GFP, LuxI and GFP both with LVA degradation tail

This study pCWqs11 pACYCB15 Plteto1-B34-LuxI- B34-GFP-B15- J23119-TetR

Cam LuxI GFP and TetR witrh LVA degradation tail

This study pCWqs12 PACYCB15 and pSB1AK3

Cm, Amp, Kan

cotransformation of s-3oc6hsl and r- 3oc6hsl

This study pCWqs13 pSB1A2 B34-

luxI.B34.GFP.B15

Amp LuxI and GFP with LVA degradation tags

Chapter 2. Backgr ound and genetic library engineering luxI.B34.GFP.B15 This study pCWqs15 pACYCB15 Ptet.B34-

luxI.B34.GFP.B15- J23119.TetR

Cam LuxI GFP and TetR with LVA

This study pCWqs16 pSB1C3 Ptet.B34-

luxI.B34.GFP.B15

Cam LuxI and GFP with LVA

This study pCWqs17 pBad33 B34-

luxI.B34.GFP.B15

Cam LuxI and GFP with LVA

This study pCWqs18 J23100 B34-GFPlva-B15-

pSB1C3

Cam GFP with LVA degradation tag;

This study pCWqs19 J23104 B34-GFPlva-B15-

pSB1C3

Cam GFP with LVA degradation tag

This study pCWqs20 J23117 B34-GFPlva-B15-

pSB1C3

Cam GFP with LVA degradation tag;

This study pCWqs21 J23107 B34-GFPlva-B15-

pSB1C3

Cam GFP with LVA degradation tag;

This study pCWqs22 pBad33 B30-sfGFP Cam sfGFP; col2

This study pCWqs23 pBad-sfGFP b30-luxI Cam LuxI with LVA tag

This study pCWqs24 B30-mCherry- pUC19

luxR.b30 Amp coding parts of 3oc6hsl receiver This study pCWqs25 B30-mCherry-

pUC19

J23104 Amp mCherry reporter

This study pCWqs26 LuxR.b30.b30- mCherry-pUC19

2.6. Genetic cir cuits built in this study 35

This study pCWqs27 luxR-pluxRI- mCherry

b30-sfGFPlva Amp sfGFP with LVA degradation tag This study pCWqs28 pBad-luxI-sfGFP b30-mCherrylva Cam luxI and mCherry with LVA tags;

colony 2

This study pCWqs29 pBad-luxI-sfGFP b30-sfGFPlva Cam luxI and sfGFP with LVA degradation tags; isolated col3

This study pCWqs30 pUC18r6k-Tn7T- Kan

b30-luxI Amp, Kan insertion of luxI sender device under Pbad control into ApaI sites

This study pCWqs31 luxR-PluxRI- sfGFP

pUC18r6k-Tn7T- Kan

Amp, Kan insertion of luxR receiver device under Plux-R/L control into ApaI sites This study pCWqs32 AraC-pBad-luxI-

mCherry

pUC19 Amp luxI and mCherry with LVA degrada-

tion tag; col5 This study pCWqs33 AraC-pBad-luxI-

sfGFP

pUC19 Amp luxI and sfGFP with LVA degradation

tag; col2 This study pCWqs34 AraC-Pbad-luxI-

sfGFP

pUC18r6k-Tn7T- Kan

Amp, Kan insertion of luxI sender device under Pbad control into ApaI sites; col23 This study pCWqs35 luxR-PluxRI-

sfGFP

pACYC184 Cam sfGFP with LVA degradation tag

This study pCWqs36 luxR-pluxRI- sfGFP

pBR322 Amp sfGFP with LVA degradation tag

This study pCWqs37 pUC19-luxR- pLpR-sfGFP

luxI Amp luxI and sfGFP with LVA degradation

tag

Chapter 2. Backgr ound and genetic library engineering

pRlux-sfGFP tion tag; synthetic receiver with luxR

strong constitutive expression; col 5

This study pCWqs40 luxI pUC19-luxR-

J23104-pRlux- sfGFP

Amp luxR, luxI and sfGFP with LVA degradation tag; synthetic receiver with strong luxR expression and luxI am- plification; col3

This study pCWqs41 luxR-pLpR-sfGFP pUC19 Amp luxR and sfGFP with LVA degrada-

tion tag; original synthetic receiver with LVA-tagged cofactor; col 3

This study pCWqs42 luxI pUC19-luxR-

pLpR-sfGFP

Amp luxR, luxI and sfGFP with LVA degradation tag; original synthetic receiver with LVA-tagged cofactor and luxI amplifier; col 4

2.6. Genetic circuits built in this study 37

In document A unified platform for experimental and computational biology (Page 59-65)