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Table 1 Strains and plasmids used in this study

From: GTP-binding protein Era: a novel gene target for biofuel production

 

Description

Reference

Strain

  

E. coli DH5α

Used for molecular cloning

[25]

S. elongatus PCC 7942

Wild-type freshwater cyanobacterium

gifted by Susan S. Golden (UCSD, California)

2A01

S. elongatus PCC 7942 containing transposon pRL1063a

This study

PCC 7942

S. elongatus PCC 7942 containing pNS3

This study

Se:era

S. elongatus PCC 7942 expressing the era gene from neutral site 3

This study

Se:Δera

S. elongatus PCC 7942 with a insertion of the inactivate transposon (pSE3) and expressing the era gene from neutral site 3

This study

Se:Δera + era

S. elongatus PCC 7942 expressing a truncated thioesterase from E. coli DH5α ('tesA) and containing a disrupted acyl-ACP synthetase (SynPCC 7942_0918)

This study

Plasmid

  

pJet1.2

Used for the cloning of blunt PCR products

Thermo-Fisher Scientific (Waltham, MA, USA)

pRL1063a

Contains a transposon based on Tn5, which bears several antibiotic resistance genes (kanamycin (Km), bleomycin (Ble) and streptomycin (Sm)), promoterless luciferase (luxAB) reporter genes, an oriV not recognized by PCC 7942 and a transposase gene. The oriT for conjugative transfer is present on the plasmid but is not part of the transposon.

[11]

pHN1-LacUV5

Targets Neutral site 3, confers resistance to chloramphenicol antibiotic (Cm) and contains a strong isopropyl-β-D-thiogalactopyranoside (IPTG)-regulated lacUV5 promoter followed by a ribosome binding site (RBS) and an unique HindIII restriction site

[15]

pNS3

Derived from pHN1-LacUV5 with the original RBS removed and lacking the ATG start codon near the multiple cloning site

This study

pNS3:ERA

Derived from pNS3 with era gene (Synpcc7942_0160) cloned into the unique HindIII restriction site behind the inducible lacUV5 promoter

This study

pSe:Δera

Inactivate transposon TN5-1063a originally isolated from strain 2A01, with the transposase mutate by digestion with NotI and blunting using T4 DNA polymerase

This study