In order to attempt to achieve the goals that we set ourselves we came up with three experiments to test our theories.
Experiment 1: Establishing Anchor Protein
1. Golden gate assembly of plasmid backbones http://synbio.tsl.ac.uk/golden-gate-assembly-protocol/
and 4 G-blocks (one of each candidate anchor protein and Zif268)
Waiting on enzymes (hopefully Friday/ Monday)
Lpp-OMPa is done
BclA, Pgsa, (INP) - waiting on enzymes to cut out anchor proteins
2. Transform electrocompetent E. Coli cells with plasmid
NB - IPTG needs to be added
Grow cells
Prepared for microscopy (mounting medium)
DNA on glass (waiting epoxy, aminated oligo, fluorescent marker) - Friday/Monday
3. Bind antibodies to flag tag - measure using fluorescent detection mechanism
Antonia has components
4. Compare intensities - select anchor protein that fluoresces brightest
Back-up:
Lyse cells to see if expression is taking place
Experiment 2: Establishing Zinc Finger DNA Binding Proteins
1. Golden gate assembly of plasmid backbones and many G-blocks (one of each candidate zinc finger protein and the winning anchor protein) and with fluorescent protein code
2. Transform E. Coli with plasmid
3. Make wells of cognate DNA of the zinc fingers, introduce transformed E. Coli to the plate, leave to bind, wash plate and compare fluorescence of the E. Coli cells to determine which zinc finger binds strongest
4. Select 4 zinc fingers which bind strongest to their cognate DNA
Ben models = requires time points, do at the same time
Microscopy - do with both alive and dead
Test both coverslip or on slide
Experiment 3: Checking Colour
1. Transform E. Coli with each colour fluorescent protein and the anchor protein and each zinc finger protein
- YFP, GFP, RFP - need to select a blue fluorescent protein
2. Test whether simple images can be made - green circle on a red square
3. Test colour mixing - mixing fluorescent cells to make colours not already present
4. Test complex images
