Example 44
Plasmids used in this study were constructed using standard cloning procedures and maintained in a KL740 strain if using an OR2-OR1 promoter (29° C.), a MG1655Z1 strain if using a Pl-tetO1 or Pl-lacO1 promoter, a BL21-DE3 strain for protein purification, a BL21 strain for promoter characterization, or a JM109 strain for all other constructs. KL740 upregulates a temperature sensitive lambda cI repressor, and MG1655Z1 upregulates tetR and lacI. PCR products were amplified using Pfu Phusion Polymerase (New England Biolabs) for all constructs except for those labeled with AlexaFluor-588-5-dUTP, which used Taq Polymerase (New England Biolabs), and were DpnI digested. Plasmids were either miniprepped using a PureYield column (Promega) or midiprepped using a NucleoBond Xtra Midi column (Macherey-Nagel). All plasmids were processed at stationery phase. Before use in the cell-free reaction, both plasmids and PCR products underwent an additional PCR purification step using a QiaQuick column (Qiagen), which removed excess salt detrimental to TX-TL, and were eluted and stored in 10 mM Tris-Cl solution, pH 8.5 at 4° C. for short-term storage and −20° C. for long-term storage.