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Distribution Kit. | Distribution Kit. | ||
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<br>09/07/2015: | <br>09/07/2015: | ||
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
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.
13/7/15
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.
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.
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
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.
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).
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).
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.
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.
Sep 07
Sep 14