User:Xi Chen:SynBioLib

General

Techniques

Gene/genome construction

SLIC/CPEC/Gibson

A nice intro from JBEI

Parts

Zinc-finger and TALE

Expected contributor: Adam

PUF

Expected contributor: Xi

Fluorescent proteins

EGFP

[DNA sequence of EGFP]

Bacteria

Techniques

Cross-cell/cross-strain/cross-species DNA transfer

Expected contributor: Brian Renda

In vivo recombination

Expected contributor: PeterE, Erik, Jiri

Parts

Replication origins

• ColE1

  To be added later (XC)

• oriS

  1 / cell, controlled by repE gene and parABC partition. -- Learned from pETcoco (Novagen)

• oriV

  40 / cell, controlled by TrfA. -- learned from pETcoco (Novagen)

Inducible promoters

• lacUV5

  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

Part organization/configuration

Impacts of spacers, orientation, promoter interferences, etc. on gene expression.

Mammalian Cells

Parts

Inducible promoters

Question: Do bacterial ones work?