Genetic Modification of Enterococcus faecalis

Nucleotide sequences of primers are listed in Table S3. All restriction enzymes were purchased from New England Biolabs. For construction of the cCF10-inducible tig complementation vector, tig was amplified from purified OG1RF genomic DNA using Pfu Ultra II polymerase (Agilent), digested with BamHI-HF/NheI-HF, and ligated to pCIEtm (23 (link)) treated with the same restriction enzymes. The plasmid construct was verified by Sanger sequencing (Eurofins). For generation of constitutive fluorescent protein constructs, P₂₃ was excised from pDL278p23 (57 (link)) by digestion with EcoRI-HF/BamHI-HF and ligated to pTCV-LacSpec digested with the same restriction enzymes. A fragment encoding promoterless GFP (58 (link)) flanked by BamHI and BlpI sites was inserted to create pP₂₃::GFP, and the BamHI-SphI fragment from pJ201::187931 was inserted to create pP₂₃::tdTomato. The Tn insertions in strains used for submerged Aclar disc and MultiRep reactor experiments were verified by colony PCR using the oligonucleotides listed in Table S4. The Tn insertion adds ∼2.1 kb to the size of the wild-type allele.

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Willett J.L., Dale J.L., Kwiatkowski L.M., Powers J.L., Korir M.L., Kohli R., Barnes A.M, & Dunny G.M. (2021). Comparative Biofilm Assays Using Enterococcus faecalis OG1RF Identify New Determinants of Biofilm Formation. mBio, 12(3), e01011-21.

Publication 2021

Pfu Ultra II polymerase Sanger sequencing

Allele Ccf10 Digestion Ecori Genomic Nucleotide sequences Oligonucleotides Pfu polymerase Plasmid Primers Protein Restriction enzymes Strains Tdtomato Vector

Corresponding Organization :

Other organizations : University of Minnesota Medical Center

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Variable analysis

independent variables

Restriction enzymes (BamHI-HF, NheI-HF, EcoRI-HF, BamHI-HF) used for digestion of DNA
Pfu Ultra II polymerase used for amplification of the tig gene
Ligation of digested DNA fragments to create plasmid constructs

dependent variables

Expression of the tig gene in the cCF10-inducible complementation vector
Expression of GFP and tdTomato fluorescent proteins in the constitutive constructs

control variables

Purified OG1RF genomic DNA used as template for tig gene amplification
PCIEtm, pTCV-LacSpec, and pJ201::187931 plasmids used as backbones for constructing the plasmid vectors

positive controls

Sanger sequencing to verify the plasmid constructs

negative controls

Not explicitly mentioned

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