Zinc-finger and TALE
Expected contributor: Adam
Expected contributor: Xi
Cross-cell/cross-strain/cross-species DNA transfer
Expected contributor: Brian Renda
In vivo recombination
Expected contributor: PeterE, Erik, Jiri
Phage as workhorse
Knockout strains and libraries
- To be added later (XC)
- 1 / cell, controlled by repE gene and parABC partition. -- Learned from pETcoco (Novagen)
- 40 / cell, controlled by TrfA. -- learned from pETcoco (Novagen)
- Engineered from wildtype lac promoters by removing cAMP regulation. Used in DE3 to drive T7 RNAP. Also said to have 2 mutations that increase activity.
- “The lambda DE3 prophage encoding T7 RNA polymerase in pET expression hosts carries the L8-UV5 promoter, which has three point mutations that distinguish it from the wild type lac promoter (Figure 1). Two point mutations in the –10 region increase promoter strength and decrease its dependence on CAP/cAMP stimulation for full activation. The third-point mutation is located in the CAP/cAMP binding site and decreases the affinity for CAP/cAMP. This mutation reduces, but does not eliminate, sensitivity to catabolite repression. The net effect of the three-point mutations is the creation of a stronger promoter that is less sensitive to the glucose effect. This allows strong IPTG induction of T7 RNA polymerase expression even in the presence of glucose.” “...supplementing LB media with glucose to a final concentration of 0.5–1.0% prevents the increased basal activity observed in cultures grown to stationary phase.” -- inNovations 13
- Note: Need summaries and literatures on transfer functions
Mobile genetic elements
Impacts of spacers, orientation, promoter interferences, etc. on gene expression.
Question: Do bacterial ones work?