Summary made by Anne Vogel
Erin K. Zess, Matthew B. Begemann, Brian F. Pfleger
Biotechnology and Bioengineering, Vol. 113, No. 2, February, 2016
Construction of New Synthetic Biology tools for the control of gene expression in the cyanobacterium Synechococcus sp Strain PCC 7002
- Paper focuses on two different methods to control gene expression: An anhydrotetracycline-dependent induction system and a trans-acting sRNA system.
Anhydrotetracycline (aTc) inducible promoters
- PEZtet promoter coupled to a TetR-dependent repression. The repression can be reversed (so expression of the gene can be induced) with the addition of aTc
- The operator is directly upstream and downstream of the -35 element, allowing for control over the 5′UTR sequence
- Compared to the IPTG induction systems, PEZtet is more tightly regulated, less “leaky” in the uninduced state and responsive to low levels of aTc, seems to have 18 fold higher expression that the Pcpt promoter used in Synechococcus 7002
- Induction at as low as 10 ng/mL and full induction at 1000 ng/mL
- aTc is light sensitive, the effect wore off after about 48h
Summary of figure 1: (A) TetR binds the tet operator (TetO) sequences (gray) near the promoter of the reporter gene: transcription initiation is blocked. Addition of aTc hinders TetR-TetO binding, thereby allowing transcription initiation (B) Design of the aTc inducible tetR promoters. The boxed -10 and -35 regions indicate the predicted elements based on the transcription start sites
The trans-acting sRNA tool
- sRNA: small regulatory RNA, can influence gene expression e.g. binding to mRNA and inducing double stranded RNA degradation
- The sRNA component of IS10 from coli was modified for use in Synechococcus PCC 7002
- sRNA called RNA-OUT: binds to the 5′ UTR of a mRNA transcript (RNA-IN), resulting in reduction of translation initiation
- Can reduce up to 70% of expression of the reporter gene (here GFP)
Summary of figure 2: When expressed, the RNA-OUT piece forms a hairpin structure that interacts with the 5′ UTR RNA-IN sequence, unfolds, and blocks translation of the reporter gene. In the absence of RNA-OUT, translation proceeds as normal.