Week 9 Lab: Cell-Free Systems

  1. Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. Name at least two cases where cell free expression is more beneficial than cell production. Describe the main components of a cell-free expression system and explain the role of each component.

  2. Why is energy provision regeneration critical in cell-free systems? Describe a method you could use to ensure continuous ATP supply in your cell-free experiment.

  3. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why. How would you design a cell-free experiment to optimize the expression of a membrane protein? Discuss the challenges and how you would address them in your setup.

  4. Imagine you observe a low yield of your target protein in a cell-free system. Describe three possible reasons for this and suggest a troubleshooting strategy for each.

what outcomes might we see at different temperatures and – if we change growht conditiosn we will see difference in gorowth and pigment productionn

OD600 – optical density at 600 wavelngth helps easure how cloudy the liquid is, to determine cell growth.

other experimental setups use acetone to separate … can do protein percipitate analysis … could also use ethanol or isopropyl alcohol to separate diferent pieces from one another …

why might we want to engineer e coli to produce pigments … because … e coli grows fast and because it does fewer cellular functions

input always influences output … everything starts with a sugar so pro … phytoene synthase … bacterial crpd rate limiting step – https://pmc.ncbi.nlm.nih.gov/articles/PMC9039723/

biosynthetic pathway knowckout experiments … knock out one gene at a time to see

which should you modify e coli to sacrromycese – maybe stick with what uis easiest to modify – also how large is your gene insert – sometimes have to put into e coli before getting into yeast – e coli grows fast and is cheaper … sacrromycese is slower and constructs may have to simplified … yeast might be better if maybe trying to modify another fungus …

chose one enzyme and outline – main peices we need when making a construct – promoter (ex. T7, conducive, on all the time), RBS (Rhibozome binding site), your coding sequence, terminator, plasmid origin of replication (tells cell to allow plasmid to copy into host), and antibiotic resistance marker