Synthetic Biology Flashcards
The forced expression of a particular gene in which the gene is usually not expressed at a desired level
Ectopic overexpression
The advent of recombinant DNA technologies
What technology ushered in the age of synthetic biology?
Any natural or synthetic system that allow initiation, interruption, or termination of target gene expression
Gene switches
What is a major drawback of constitutive expression systems?
lack of precise control over spatiotemporal expression and activity of proteins
What is needed if synthetic biology is going to be used in human therapies?
very tight control of the activity of foreign genetic elements
Which synthetic biology technology is achieving unprecedented success in human clinical trials?
CAR-T cell therapy to treat blood cancers ( solid tumors still prove recalcitrant)
What is the current focus of synthetic biology in mammalian systems?
designing synthetic gene circuits consisting of interconnected switches to program time dependent and context dependent target gen activities in living cells
gene switches acting at the transcriptional level are operated by:
Trans regulators consisting of sequence-specific DNA binding domain fused to an epigenetic regulator
What is a transregulator?
A chimeric regulatory protein consisting of a trafficking domain(to target DNA, RNA, protein) and a regulatory domain(specifies target activity)
What should an optimal inducible gene switch do?
permit essentially no expression of target gene in absence of trigger signal, while enabling maximal expression in activated state
What are some conventional approaches to engineer stimulus responsive gene expression in human cells?
1) systematic use of ligand-responsive prokaryotic TFs as DBD of eukaryotic trans-regulators 2) Engineering transcriptional gene switches responding to stimuli delivered to cell surface receptors 3)RNA level gene switches
What is the tighest transgene developed to date?
The LTRi switch, developed by Jim Collins in 2007
Describe how the LTRi circuit works
The LacI-TetR-RNAi switch works as follows:
In OFF state, transgene expression is abolished by LacI dependent transcription repression AND cocomitant RNAi by short hairpin RNA(shRNA) that targets the 3’UTR of the transgene mRNA.
Addition of IPTG de-repressed LacI specific promotors, resulting in transgene transcription and concomitant TetR mediated repression of shRNA production
How could one make a ligand responsive gene switch?
Could use an RNA aptamer- RNA aptamers undergo significant conformational change when bound to protein-as such, ligand responsive aptamers could be incorporated into different RNA regions to create inducible gene switches
How could one capture pathway-specific endogenous signalling events with user-defined transgene readouts?
Receptor mediated signalling cascades activate specific endogenous promotors; engineering of synthetic promotors containing the same response elements as the endogenous promotor creates a virtual clone on an episomal vector
What are two ways could one design trigger inducible degradation of a target protein?
1) Forced recruitment of a binding partner that contains a conditional degron, which drives proteasomal degradation
2) Proteasome independent control of protein degradation can be achieved by incorporating cleavage sites for contionally activated TEV protease into a target protein
