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Table 1 Cloning parts, plasmids, strains and primers used in this study

From: Ethylene production with engineered Synechocystis sp PCC 6803 strains

Cloning parts, plasmids, strains and primers

Description

Source/remark

RBS a

  

Rbs-O

AGGAGGACTAGCATG

[2]

Rbs-34

ATTAAAGAGGAGAAAACTAGCATG

[15]

Rbs-30

AAAGAGGAGAAAACTAGCATG

[15]

Rbs-H

TAGTGGAGGTACTAGCATG

RBS* from [15]

Rbs-C9

AAAGGAGGTGATAGCATG

This study

Rbs-C10

AAAGGAGGTGATTAGCATG

This study

Rbs-C11

AAAGGAGGTGATCTAGCATG

This study

Promoter

  

Ptrc

TTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACAC

[2]

3 × Ptrc

TTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACA

This study

5 × Ptrc

TTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACA

This study

Terminator

  

tt

Same as B0014 from http://www.partsregistry.org

[2]

Plasmids

  

pHKH001

Integration vector disrupting slr0168 in Synechocystis genome with kanamycin resistance cassette a selection marker

[2]

pHKH_BB

Bio-brick compatible derivative of pHKH001 carrying the Ptrc promoter and tt-transcription terminator

This study

pHKH_rbsO_efe

Integration vector carrying efe under Ptrc promoter and Rbs-O

This study

pHKH_rbs34_efe

Integration vector carrying efe under Ptrc promoter and Rbs-34

This study

pHKH_rbs30_efe

Integration vector carrying efe under Ptrc promoter and Rbs-30

This study

pHKH_rbsH_efe

Integration vector carrying efe under Ptrc promoter and Rbs-H

This study

pHKH_rbsc9_efe

Integration vector carrying efe under Ptrc promoter and Rbs-C9

This study

pHKH_rbsc10_efe

Integration vector carrying efe under Ptrc promoter and Rbs-C10

This study

pHKH_rbsc11_efe

Integration vector carrying efe under Ptrc promoter and Rbs-C11

This study

pHKH_3xPtrc_efe

Integration vector carrying efe under 3xPtrc promoter and Rbs-30

This study

pHKH_5xPtrc_efe

Integration vector carrying efe under 5x Ptrc promoter and Rbs-30

This study

pVZ_LS

A bio-brick compatible version of mobilizable broad host self-replication plasmid pVZ321 containing the RSF1010 replicon, and carrying the Ptrc promoter, Rbs30 and tt-transcription terminator

Plasmid pVZ321 from [42]

pVZ-efe

pVZ plasmid carrying efe gene under Ptrc promoter and rbs 30

This study

pVZ_Nhis-efe

pVZ plasmid carrying N-terminal hexa-histidine tagged efe under promoter Ptrc and Rbs-30

This study

Strains

  

Wild-type (WT)

Synechocystis sp. PCC6803, glucose tolerant, naturally transformable

Bhaya (Stanford)

VPV1

WT, Slr0168::Ptrc-rbsO-efe-Kmr

This study

VPV2

WT, Slr0168::Ptrc-rbs34-efe-Kmr

This study

VPV3

WT, Slr0168::Ptrc-rbs30-efe-Kmr

This study

VPV4

WT, Slr0168::Ptrc-rbsH-efe-Kmr

This study

VPV5

WT, Slr0168::Ptrc-rbsc9-efe-Kmr

This study

VPV6

WT, Slr0168::Ptrc-rbsc10-efe-Kmr

This study

VPV7

WT, Slr0168::Ptrc-rbsc11-efe-Kmr

This study

VPV40

WT, Slr0168::3xPtrc-rbsO-efe-Kmr

This study

VPV43

WT, Slr0168::5xPtrc-rbsO-efe-Kmr

This study

SAW11

Glycogen knock out strain

[36]

VPV55

WT carrying plasmid pVZ _efe

This study

VPV56

SAW11 carrying plasmid pVZ _efe

This study

VPV58

SAW11 carrying plasmid pVZ_Nhis_efe

This study

VPV62

WT carrying plasmid pVZ_Nhis_efe

This study

VPV65

SAW11, Slr0168::Ptrc-rbs30-efe-Kmr

This study

Primers

  

efe-bb-fwd

GCGGAATTCGCGGCCGCTTCTAGAGGAGGACTAGCATGACCAACTTGCAAACCTTTGAAT

 

efe-bb-r

GTACTGCAGCGGCCGCTACTAGTATTAGGAGCCGGTGGCGCG

 

vpv-1-st-Hind-R

CTAGTAAAGCTTATCAATACTTTCCACCCC

 

VPV-efe-Bamh1-f-noatg

CCATCACGGATCCACCAACTTGCAAACCTTTGAATTGC

 

VPV-efe-st-Hind3-rev

TAATTAAGCTTATCAGGAGCCGGTGGCGCGGGTATCGG

 

V-rbs30-efe-f

GACAGCTAGCATTAAAGAGGAGAAAACTAGCATGACCAACTTGCAAACC

 

V-rbs34-efe-f

GACAGCTAGCAAAGAGGAGAAAACTAGCATGACCAACTTGCAAACC

 

V-rbsC10-efe-f

GACAGCTAGCAAAGGAGGTGATTAGCATGACCAACTTGCAAACC

 

V-rbsC11-efe-f

GACAGCTAGCAAAGGAGGTGATCTAGCATGACCAACTTGCAAACC

 

V-rbsC9-efe-f

GACAGCTAGCAAAGGAGGTGATAGCATGACCAACTTGCAAACC

 

V-rbsH-efe-f

GACAGCTAGCTAGTGGAGGTACTAGCATGACCAACTTGCAAACC

 

v-bbs-pUC-r

CGGGATCCGATCCAATCTGCAGCGGCCGCTACTAGTA

 

H1-SEG-VF

TGTCGCCGCTAAGTTAGACCGC

 

H2-SEG-VR

CTGTGGGTAGTAAACTGGCAATGCC

 

H1-SEQ-VF

CGGCAATGGTCCCAAAAT

 

KanF-SEQ-VR

AGACGTTTCCCGTTGAAT

 

pVZ321-SEQ-F

CGCAGGGCTTTATTGATT

 

pVZ321-SEQ-R

CCCCCCCCACTCTATTGTA

 

efe-n–H-f

CAGATATGACATATGCATCATCATCATCATCATACCAACTTGCAAACCTTTGAATTGC

 

efe-ba-r

ACCTAGGTCAGTAGGATCCTTATTAGGAGCCGGTGGCGCGGGTATCG

 
  1. a RBS sequence underlined and start codon ATG is shown in bold