Plasmid Construction for Fluorescent Reporters

Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (Ipswich, MA). PCR was performed using Herculase DNA polymerase (Stratagene). PCR primers were obtained from Integrated DNA Technologies. All the plasmids used in this study are derivatives of p2TK2-SW2 mCh(Gro) (Agaisse and Derre, 2014 (link)). They express mCherry under the control of the groESL operon promoter and terminator, the TetR repressor and the indicated Inc under the control of the tetA gene promoter and incDEFG operon terminator. The p2TK2-SW2 mCh(Gro) Tet IncD-, IncE-, and IncG-3xFLAG plasmids were described previously (Agaisse and Derre, 2014 (link); Mirrashidi et al., 2015 (link)). p2TK2-SW2 mCh(Gro) Tet IncD-Myc, CTL0314-3xFLAG, CTL0475-3xFLAG, or IncD/IncE-3xFLAG chimera were constructed similarly using the primers listed in Supplementary Table S1.

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Han Y, & Derré I. (2017). A Co-infection Model System and the Use of Chimeric Proteins to Study Chlamydia Inclusion Proteins Interaction. Frontiers in Cellular and Infection Microbiology, 7, 79.

Publication 2017

T4 DNA ligase PCR primers

Chimera Derivatives Dna polymerase Gene control Operon Plasmids Primers Restriction enzymes T4 dna ligase Teta

Corresponding Organization : University of Virginia

Other organizations : Yale University

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Variable analysis

independent variables

Restriction enzymes and T4 DNA ligase obtained from New England Biolabs (Ipswich, MA)
PCR performed using Herculase DNA polymerase (Stratagene)
PCR primers obtained from Integrated DNA Technologies
P2TK2-SW2 mCh(Gro) plasmid and its derivatives expressing mCherry, TetR repressor, and indicated Inc under the control of specific promoters and terminators

dependent variables

Not explicitly mentioned

control variables

All the plasmids used in this study are derivatives of p2TK2-SW2 mCh(Gro) (Agaisse and Derre, 2014)

positive controls

P2TK2-SW2 mCh(Gro) Tet IncD-, IncE-, and IncG-3xFLAG plasmids (Agaisse and Derre, 2014; Mirrashidi et al., 2015)

negative controls

Not explicitly mentioned

Annotations

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