Team:Warwick/Project5
Week 1
We brainstormed ideas of what we can do with the project, more specifically and also split into smaller groups of modellers and biologists (although we had daily meetings). We spent a lot of time coming up with and optimising DNA sequences for zinc fingers.
Jun 29
Week 2
07/07/2015:
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Agar plates (with chloramphenicol and streptomycin) made.
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Top 10 cells made electrocompetent.
08/07/2015:
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Transformed Top 10 cells with INP, Lpp OmpA and Zif 23 from 2015 DNA
Distribution Kit.
09/07/2015: -
Mini-prepped cells from yesterday. -
Nanodrop results for mini-prepped plasmids.
Ran an electrophoresis gel with plasmids from the mini-prep. Jul 06
Week 3
13/07/2015:
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Grew up MG1655-Z1 cells (added Streptomycin).
14/07/2015:
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Made MG1655-Z1 cells competent.
Week 4
22/07/2015:
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Ligated full construct gBlock into pSB1C3 plasmid backbone.
23/07/2015:
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Transformed ligated plasmid into Top 10 cells.
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Ran PCR of gBlocks.
24/07/2015:
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Transformed ligated plasmid into Top 10 cells again.
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Ran PCR of BclA, INP and pgsA gBlocks.
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Transformed more cells with ligated plasmid (using electroporation).
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Plated transformed cells.
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Ran PCR of sZF2, sZF10 and sZF14.
Week 5
27/07/2015:
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Chemically transformed plates and electrotransformed plated grew over
weekend.
28/07/2015
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Colony PCR of the 2 clones.
29/07/2015
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Conducted plasmid mini prep of bacterial cultures.
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Ran gel electrophoresis of redone PCR.
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Ran gel electrophoresis of mini prep restriction digestion.
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Redoing PCR (of INP, sZF2, sZF10 and sZF14) with DMSO (at 0%, 3% and
6%).
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PCR purification of BclA and and pgsA.
30/07/2015
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Re-did PCR of INP, sZF10 and sZF14.
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sZF14 returned positive (concentration of 31.8 ng/µl after purification).
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Purified sZF2.
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Re-doing PCR of INP and sZF10.
31/07/2015
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Gel of sZF10 and INP PCR product returned negative.
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Re-doing PCR with 66 ̊ C and 68 ̊ C annealing temperatures.
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Ran gradient PCR of sZF10 and INP.
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Did restriction digestion of BclA, pgsA and Lpp OmpA
Week 6
03/08/2015:
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Re-did restriction digestion of Lpp OmpA.
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Ligated BclA and pgsA into pSB1C3.
04/08/2015:
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Transformed ligated plasmid into MG1655-Z1 cells.
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PCR purified INP.
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Did a restriction digestion of INP and Lpp OmpA.
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Ran a gradient PCR of Lpp OmpA full construct.
05/08/2015:
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2 cultures inoculated with colonies from a DH5α Z1 plate.
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Purified and restriction digested Lpp OmpA full construct.
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Restriction digestion of INP.
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Streaked plate with pgsA and BclA transformed cells.
06/08/2015:
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Gel electrophoresis of sZF10.
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Cut out band corresponding to sZF10 (at 270 bases).
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Did a gel extraction (using Freeze ‘n’ Squeeze method).
07/08/2015:
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Streaked chloramphenicol plates with transformed RFP DH5α Z1 cells.
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Re-streaked plates with BclA and pgsA transformed cells.
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Made chemically competent DH5 α Z1 cells.
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Ran gel of digested plasmid. Obtained single band at 2000 base pairs (where
backbone without RFP would be, lacking band at 1000 base pairs (for RFP gene)).
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Ran gel of undigested plasmids, single band at 3000 base pairs.
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Gel purified digested plasmid, nanodropped (2.1ng/l).
08/08/2015:
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RFP and BclA transformed cells grew with single colonies.
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pgsA plate showed no growth.
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Inoculated falcon tubes with RFP and BclA.
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Inoculated tube using old pgsA.
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Plated pure DH5α Z1 onto 1 chloramphenicol and 1 clean plate (to establish
background growth).
09/08/2015:
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Mini prepped RFP, BclA and pgsA transformed cells.
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Chloramphenicol plate grew no colonies, clean plate grew a lawn.
Week 7
10/08/2015:
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Nanodropped mini prep of RFP, BclA and pgsA.
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Sent off for sequencing.
11/08/2015
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Digested old EC1 plasmid using Pst1+EcoR1 and Age1+Nde1 (to obtain
plasmid backbone, full construct and anchor proteins).
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Digested new RFP plasmid to obtain plasmid backbone.
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Inoculated culture with RFP bacteria.
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PCR of sZF10.
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Mini-prep of RFP plasmid (JO4450).
12/08/2015
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Prepared 2 binding glass slides and 1 control.
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Mini prepped new RFP plasmid.
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Digested new RFP plasmid and ran it on a gel.
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Gel of old full construct digested by Age1+Pst1.
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Gel extracted then nanodropped. Showed that the gel extraction didn’t work.
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Digestion of all zinc fingers.
13/08/2015
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Ligation of Lpp OmpA full construct into pSB1C3backbone.
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Transformation of ligation product.
14/08/2015
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Restriction digestion of Lpp OmpA full construct using Pst1+EcoR1.
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Inoculation of Lpp OmpA full construct transformed plasmid.
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Only 2 colonies on the 4 plates of bacteria grew (background growth is 0 via
control plates).
15/08/2015
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Mini-prepped grown transformed DH5α Z1, colonies 1 and 2.
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Plated normal DH5α Z1 on clean plates (found a lawn of viable cells).
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Re-transformed cells formed a lawn (taken to be re-spread and inoculated).
Week 8
17/08/2015:
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Prepared mini prep for sequencing.
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Digested Lpp OmpA out of the plasmid and gel extracted the backbone.
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Ligated all anchor proteins into the plasmid.
18/08/2015:
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Transformed DH5α Z1 cells with each anchor protein.
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Re-growing Lpp OmpA transformed DH5α Z1 cells.
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Electroporated MG1655Z1 cells using the original full construct plasmid and
plated.
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Streaked FIM(negative) cells on a clean plate.
19/08/2015:
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All plated grew well (Lpp OmpA transformed cells formed a lawn).
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Streaked Lpp OmpA transformed cells onto a chloramphenicol plate.
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Electroporated cells did not grow.
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Inoculations were made of the BclA, pgsA and INP plates.
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Made 12 new chloramphenicol plates.
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Designed primers (for the DNA origami oligonucleotide adhesive) for the
modellers.
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Sequences came back clean (with no mutations).
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Colony PCR of all grown cells.
20/08/2015:
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Ran an electrophoresis gel of the colony PCRs.
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Made chemically competent FIM(negative).
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Made glycerol stocks of all completed cell cultures.
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Mini prep of pgsA, BclA and INP plasmids.
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Made inoculations of Lpp OmpA (one with IPTG, one without, and one where
IPTG is added later).
21/08/2015:
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BclA, INP and pgsA plasmids sent for sequencing.
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Finished making FIM(negative) chemically competent cells.
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Measured OD600 of Lpp OmpA inoculations.
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Made another Lpp OmpA inoculation.
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Remade the colony PCR.
22/08/2015:
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Ran electrophoresis gel of colony PCR.
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No results so colony PCR remade.
23/08/2015
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No results.
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Made inoculations of Lpp OmpA transformed cells (with IPTG and normal DH5α
Z1 cells).
Week 9
24/08/2015:
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Made colony PCR again using different primer-colony combinations.
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Made more chloramphenicol plates.
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Assembled primers.
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Made designs (using paper stencils) of RFP transformed DH5α Z1.
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Carried out Age1+Nde1 digestion of Lpp OmpA full construct.
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Ran electrophoresis gel of colony PCR – had negative results.
25/08/2015:
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Gel extraction of digested full construct plasmid showed no positive results.
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Mini prepped functional cell.
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Set up (8 hour) digestion.
26/08/2015:
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Analyse digestion. Showed a single band (a negative result).
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Put DH5αcells on to grow (for microscopy).
27/08/2015:
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First steps of slide preparation.
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Mini prepped Lpp OmpA full construct.
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Digested Lpp OmpA full construct with Age1.
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Ran an electrophoresis gel of the digestion - results were negative.
28/08/2015:
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Mini prepped Lpp OmpA full construct.
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Ran electrophoresis gel (of digestion with EcoR1+Nde1 and digestion with
EcoR1+Age1).
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Rerunning electrophoresis gel (using old enzymes and Lpp OmpA plasmid full
construct mini prep).
29/08/2015:
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Did gel extraction of Lpp OmpA full construct plasmid mini prep.
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No supernatant after freeze and squeeze step.
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MG1655Z1 cells transformed with PSB3K3 via electroporation.
30/08/2015:
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Redid gel extraction of Lpp OmpA full construct (plasmid mini prep) via.
Week 10
31/08/2015:
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Repeated gel extraction (not enough full construct mini prepped plasmid).
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Started mini prep of Lpp OmpA full construct transformed colony.
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Slide prep up to (but not including) Blocking Buffer wash.
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LB inoculated with colony from MG1655Z1 + pSB3K3 plate.
01/09/2015
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Gel extracted anchor protein plasmids (from digestion).
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Microscopy session - some controls not the best, but significant difference in
brightness of induced and Lpp OmpA full construct.
02/09/2015:
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PCR of sZFs 2, sZF10 sZFand 14.
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Mini prepped more plasmid from the working MG1655Z1cells.
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Set up more MG1655Z1cells to grow overnight.
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Restriction digestion of full construct plasmid carried out using Nhe1+Cla1.
03/09/2015:
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Triplicate mini prep made using colony 2 (MG1655Z1).
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Restriction digested mini prep.
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Ran a gel extraction of digestion, then extracted from gel.
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Ran a PCR of the cut zinc fingers.
04/09/2015:
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Mini prepped a large batch of Dh5α Z1cell.
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Ran a restriction digestion (using Age1+Nde1).
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JS006 transformed with PSB3K3 via electroporation, grown on 1:2000
Kanamycin plate.
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Successful PCR of sZF2 and sZF14 (purified and tested).
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Redid PCR of sZF10.
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Gel extraction of plasmid Mhe1+Cla1 digest, worked perfectly.
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PCR purified the 3 anchor proteins (BclA, pgsA and INP).
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Mini prepped anchor proteins.
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Digested the anchor proteins, ran on an electrophoresis gel and then extracted.
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Carried out a restriction digestion (with Age1+Nde1).
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Ran digestion on an electrophoresis gel, and then extracted.
05/09/2015
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Gel extracted the plasmid digested by Nhe1+Cla1.
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Mini prepped more plasmid.
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Digestion of plasmid (using Nde1+Age1).
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Ran electrophoresis gel of Nde1+Age1 digestion.
Week 11
07/09/2015:
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Ran an electrophoresis gel of Nde1+Age1 digest (reran to separate out bands).
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Gel extracted.
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Digested all zinc fingers.
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Gel of full construct (digested with Nhe1+Cla1) showed a positive result, so PCR
purified.
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PCR purified sZF2, sZF10 and sZF14.
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Ligated (using T4 DNA ligase protocol).
08/09/2015:
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Ran gel electrophoresis of ligated zinc fingers (to check them).
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Transformed some of the ligation into chemically competent DH5α Z1 cells.
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Transformed rest of the ligation into electrocompetent cells.
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Made more chloramphenicol plates.
09/09/2015:
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Primer assembly of fluorescent zinc finger specific binding sequences.
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Made oligonucleotides for glass DNA binding.
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Made fluorescent oligos for specific DNA binding test.
10/09/2015:
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Plasmid preparation of pSB3K3 for insertion of fluorescent gBlocks.
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Wells 6F/18A/18C/18E/18E from kit Plate 4 2015 Phusion PCR.
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Gel electrophoresis of PCR products - showed only primer dimer at 100 bp.
11/09/2015:
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Plasmid preparation of pSB3K3 for insertion of fluorescent gBlocks.
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Wells 6F/18A/18C/18E/18E from kit Plate 4 2015 Phusion PCR.
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Gel electrophoresis of PCR products - showed only primer dimer at 100 base
pairs again.
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Mini prepped electroporated MG1655Z1 containing sZFf2, sZF10, sZF14, BclA
and INP plasmids.
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Sent the 5 modified plasmids for sequencing.
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Received PCR product from Oxford iGEM. Assembled oligos in PCR machine.
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Prepared slides (of induced and uninduced zinc fingers) for microscopy using
Dh5α Z1 cells.
12/09/2015:
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Made inoculations of MG1655Z1 transformed cells for preparation of slides
(using induced and uninduced zinc fingers and anchors).
Final Week
14/09/2015
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Redo Phusion PCR (gradient) of wells 6F/18A/18C/18E/18E from kit Plate 4
2015 with +/- DMSO.
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Gel electrophoresis of PCR products identified band of 2.5kb.
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Dpn1 digest for 3 hours and 37°C
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Remade glass slides with all zinc fingers (for the oligo binding experiment –
experiment 3) and anchor proteins (for FLAG tag experiment – experiment 2).
15/09/2015
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PCR purification of 18C and 18E.
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Gibson assembly of 18C and 18E, each with the gBlock for mKate, GFP and
Cerulean
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Remade slides with zinc fingers (for experiment 3) and anchor proteins
(experiment 2). Found out later that sequencing did not come back right, so slides
must be remade using new cells. .
16/09/2015
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Grew up all zinc fingers cells in m9 (to be used for experiment 2).
17/09/2015
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Cells had not grown up, so regrew them in LB.
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Once grown, these cells were used to prepare slides for experiment 2.
18/09/2015
Visualised slides of zinc finger transformed cells, but only got negative results.
Remade slides using alternative protocol (did not fix cells to slide, cells were in liquid media). Visualised, got positive results for sZF14.