"

From 2014.igem.org

Colonies from Gibson of pBbE1a backbone- CsgA- 48 Linkiner- SynZIP1 did not grow Error amplifying the backbone HBD 5 and 7 were used as primers (point inward towards CsgA) Primers usually used for sequencing HBD 106 and 107 are the correct primers They amplify the entire backbone, just linearize it with a cut after the flag tag Amplified the linear backbone with HBD 106 and 107 primers

Amplified the gblock - SpyTag with the stop codon included this time

Gibsoned the SpyTag and SynZIP1, SynZIP4 and SynZIP5 into their respective linearized pBbE1a backbone SpyTag→ 48 linker backbone (without flag tag) SynZIPs → 48 linker backbone with the flag tag

Transformed SpyTag plasmid and SynZIP plasmids into Turbo cells using the heat shock protocol