Week 1 (04/07 – 08/07)
Overview of molecular biology, CRISPR, cloning/plasmids, RNA design
Primers for Gibson assembly of GFP-dCAS9 in pSB1C3 designed and ordered
1st batch of competent cells (DH5α) prepared
Transformed interlab plasmids into DH5alpha competent cells
Week 2 (11/07 – 15/07)
GFP and dCas9 was amplified by PCR and extracted
Prepared more competent cells (Top10)
Issues with low PCR yield addressed by testing with gradient PCR
Gibson assembled and transformed first construct (GFP-dCas9) into Top10 E.coli
Week 3 (18/07 – 22/07)
Interlab samples were transformed into our electrocompetent Top10 cells
Colony PCR of first construct failed twice but restriction digest was successful
Colonies that showed highest uptake of constructed plasmid were sent for sequencing and stabilates prepared.
Sequencing data showed one colony had no mutations present in the required construct
Made up additional antibiotic plates (ampicillin, tetracycline, kanamycin)
Various biobrick plasmids obtained and transformed into Top10 cells.
Week 4 (25/07 – 29/07)
More primers arrived so we could begin constructing components that need to go into our second plasmid.
Plasmids amplified by PCR with Golden gate or Gibson primers to form backbones. T7 RNAP was also amplified. It took a few attempts for the amplification to succeed with both Gibson and Goldengate.
Interlab study continued
Week 5 (01/08 – 05/08)
GFP-dCas9 assembled into pSB4A5, transformed into Top10 cells and grown up.
Colony PCR of eight of the colonies was completed – showed negative results for all colonies
Colony PCR of sixteen new colonies was completed - continued to show negative results
Different fusion protein sequences amplified – all successful (no Ms2-sigma or any of the T7's at this stage)
Week 6 (08/08 – 12/08)
Protein fusion sequences ligated into pSB3T5 backbone, transformed into Top10 cells and grown up.
RpoZ competent cells produced
Digest set up for GFP-dCas9 in pSB1C3
Week 7 (15/08 – 19/08)
olony PCR of plasmid 2 constructs undertaken – partially successful
Glycerol stocks of successful colonies produced and sent for sequencing
Week 8 (22/08 – 26/08)
There was at least one colony that came back successfully from sequencing for each of the following; Com-omega, Ms2-Omega, Ms2-sigma54, Pp7-omega and Pp7-sigma54
The five plasmid 2 inserts above were digested successfully and extracted
Amplified pSB3T5 backbone
Week 9 (29/08 – 02/09)
First extraction of the five plasmid 2 inserts failed so the process was repeated from the original glycerol stocks – successful
Attempted to amplify sgRNA – Partially successful
The five plasmid 2 inserts were digested (EcoRI/SpeI)
pSB3T5 and pSB1C3 digested (EcoRI/SpeI) and
Ligation reaction set up to insert the five plasmid 2 constructs into pSB1C3 backbone
PCR of sgRNA cassette – successful
Week 10 (05/09 – 09/09)
Successfully cotransformed Top10 cells with pSB1C3/pSB3T5, pSB1C3/pSB4A5, pSB3T5/pSB4A5 and pSB1C3/pSB3T5/pSB4A5 also successfully transformed with sgRNA in pSB1C3
PheA terminator successfully added to the 3' end of the GFP in the GFP-dCas9 construct in the pSB1C3 backbone.
Week 11 (12/09 – 16/09)
GFP-PheA-dCas9 digested with (EcoRI/SpeI) and successfully ligated into pSB4A5 backbone
PCR of sgRNA and pSB1C3 extracted and Gibson assembled
Week 12 (19/09 – 23/09)
Amplified and assembled PAM staggered sequence into GFP-PheA-dCas9 in pSB1C3
Cloned PAM-GFP-PheA-dCas9 into pSB4A5
Week 13 (26/09 - 30/09)
Assembled different promoters for insertion into the PAM-GFP-PheA-dCas9 construct
Made multiple attempts to insert promoters
Week 14 (03/10 - 07/10)
Insertion of promoters partially successful
Attempts made to insert fusion proteins (e.g. MS2-sigma) into pSB1C3
Week 15 (10/10 - 14/10)
Created a library of sgRNA targeting regions by annealing complementary primers
Golden gate assembled library into sgRNA construct in pSB1C3
Transformed cells with PP7-omega in pSB3T5, and PAM-omegaProm-GFP-PheA-dCas9 in pSB4A5 in preparation for insertion of sgRNA in pSB1C3.
Transformed prepared cells with library.
Week 16 (17/10 - 21/10)
Cloned versions of our fusion proteins into PSB1C3
Tested a full construct version of our device with PP7 omega, PAM-omegaProm-GFP-PheA-dCas9, library-sgRNA in order to determine whether fluorescence difference was significant between constructs including sgRNAs targeting the PAMs and constructs that include sgRNAs with no target site.
