Difference between revisions of "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: -
Agar plates (with chloramphenicol and streptomycin) made. -
Top 10 cells made electrocompetent.
08/07/2015: -
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

13/7/15
13/07/2015: -
Grew up MG1655-Z1 cells (added Streptomycin).
14/07/2015: -
Made MG1655-Z1 cells competent.

Jul 13

Week 4

22/07/2015: -
Ligated full construct gBlock into pSB1C3 plasmid backbone.
23/07/2015: -
Transformed ligated plasmid into Top 10 cells. -
Ran PCR of gBlocks.
24/07/2015: -
Transformed ligated plasmid into Top 10 cells again. -
Ran PCR of BclA, INP and pgsA gBlocks. -
Transformed more cells with ligated plasmid (using electroporation). -
Plated transformed cells. -
Ran PCR of sZF2, sZF10 and sZF14.

Jul 20

Week 5

27/07/2015: -
Chemically transformed plates and electrotransformed plated grew over weekend.
28/07/2015 -
Colony PCR of the 2 clones.
29/07/2015 -
Conducted plasmid mini prep of bacterial cultures. -
Ran gel electrophoresis of redone PCR. -
Ran gel electrophoresis of mini prep restriction digestion. -
Redoing PCR (of INP, sZF2, sZF10 and sZF14) with DMSO (at 0%, 3% and 6%). -
PCR purification of BclA and and pgsA.
30/07/2015 -
Re-did PCR of INP, sZF10 and sZF14. -
sZF14 returned positive (concentration of 31.8 ng/µl after purification). -
Purified sZF2. -
Re-doing PCR of INP and sZF10.
31/07/2015 -
Gel of sZF10 and INP PCR product returned negative. -
Re-doing PCR with 66 ̊ C and 68 ̊ C annealing temperatures. -
Ran gradient PCR of sZF10 and INP. -
Did restriction digestion of BclA, pgsA and Lpp OmpA

Jul 27

Week 6

03/08/2015: -
Re-did restriction digestion of Lpp OmpA. -
Ligated BclA and pgsA into pSB1C3.
04/08/2015: -
Transformed ligated plasmid into MG1655-Z1 cells. -
PCR purified INP. -
Did a restriction digestion of INP and Lpp OmpA. -
Ran a gradient PCR of Lpp OmpA full construct.
05/08/2015: -
2 cultures inoculated with colonies from a DH5α Z1 plate. -
Purified and restriction digested Lpp OmpA full construct. -
Restriction digestion of INP. -
Streaked plate with pgsA and BclA transformed cells.
06/08/2015: -
Gel electrophoresis of sZF10. -
Cut out band corresponding to sZF10 (at 270 bases). -
Did a gel extraction (using Freeze ‘n’ Squeeze method).
07/08/2015: -
Streaked chloramphenicol plates with transformed RFP DH5α Z1 cells. -
Re-streaked plates with BclA and pgsA transformed cells. -
Made chemically competent DH5 α Z1 cells. -
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)). -
Ran gel of undigested plasmids, single band at 3000 base pairs. -
Gel purified digested plasmid, nanodropped (2.1ng/l).
08/08/2015: -
RFP and BclA transformed cells grew with single colonies. -
pgsA plate showed no growth. -
Inoculated falcon tubes with RFP and BclA. -
Inoculated tube using old pgsA. -
Plated pure DH5α Z1 onto 1 chloramphenicol and 1 clean plate (to establish background growth).
09/08/2015: -
Mini prepped RFP, BclA and pgsA transformed cells. -
Chloramphenicol plate grew no colonies, clean plate grew a lawn.

Aug 3

Week 7

10/08/2015: -
Nanodropped mini prep of RFP, BclA and pgsA. -
Sent off for sequencing.
11/08/2015 -
Digested old EC1 plasmid using Pst1+EcoR1 and Age1+Nde1 (to obtain plasmid backbone, full construct and anchor proteins). -
Digested new RFP plasmid to obtain plasmid backbone. -
Inoculated culture with RFP bacteria. -
PCR of sZF10. -
Mini-prep of RFP plasmid (JO4450).
12/08/2015 -
Prepared 2 binding glass slides and 1 control. -
Mini prepped new RFP plasmid. -
Digested new RFP plasmid and ran it on a gel. -
Gel of old full construct digested by Age1+Pst1. -
Gel extracted then nanodropped. Showed that the gel extraction didn’t work. -
Digestion of all zinc fingers.
13/08/2015 -
Ligation of Lpp OmpA full construct into pSB1C3backbone. -
Transformation of ligation product.
14/08/2015 -
Restriction digestion of Lpp OmpA full construct using Pst1+EcoR1. -
Inoculation of Lpp OmpA full construct transformed plasmid. -
Only 2 colonies on the 4 plates of bacteria grew (background growth is 0 via control plates).
15/08/2015 -
Mini-prepped grown transformed DH5α Z1, colonies 1 and 2. -
Plated normal DH5α Z1 on clean plates (found a lawn of viable cells). -
Re-transformed cells formed a lawn (taken to be re-spread and inoculated).

Aug 10

Week 8

17/08/2015: -
Prepared mini prep for sequencing. -
Digested Lpp OmpA out of the plasmid and gel extracted the backbone. -
Ligated all anchor proteins into the plasmid.
18/08/2015: -
Transformed DH5α Z1 cells with each anchor protein. -
Re-growing Lpp OmpA transformed DH5α Z1 cells. -
Electroporated MG1655Z1 cells using the original full construct plasmid and plated. -
Streaked FIM(negative) cells on a clean plate.
19/08/2015: -
All plated grew well (Lpp OmpA transformed cells formed a lawn). -
Streaked Lpp OmpA transformed cells onto a chloramphenicol plate. -
Electroporated cells did not grow. -
Inoculations were made of the BclA, pgsA and INP plates. -
Made 12 new chloramphenicol plates. -
Designed primers (for the DNA origami oligonucleotide adhesive) for the modellers. -
Sequences came back clean (with no mutations). -
Colony PCR of all grown cells.
20/08/2015: -
Ran an electrophoresis gel of the colony PCRs. -
Made chemically competent FIM(negative). -
Made glycerol stocks of all completed cell cultures. -
Mini prep of pgsA, BclA and INP plasmids. -
Made inoculations of Lpp OmpA (one with IPTG, one without, and one where IPTG is added later).
21/08/2015: -
BclA, INP and pgsA plasmids sent for sequencing. -
Finished making FIM(negative) chemically competent cells. -
Measured OD600 of Lpp OmpA inoculations. -
Made another Lpp OmpA inoculation. -
Remade the colony PCR.
22/08/2015: -
Ran electrophoresis gel of colony PCR. -
No results so colony PCR remade.
23/08/2015 -
No results. -
Made inoculations of Lpp OmpA transformed cells (with IPTG and normal DH5α Z1 cells).

Aug 17

Week 9

24/08/2015: -
Made colony PCR again using different primer-colony combinations. -
Made more chloramphenicol plates. -
Assembled primers. -
Made designs (using paper stencils) of RFP transformed DH5α Z1. -
Carried out Age1+Nde1 digestion of Lpp OmpA full construct. -
Ran electrophoresis gel of colony PCR – had negative results.
25/08/2015: -
Gel extraction of digested full construct plasmid showed no positive results. -
Mini prepped functional cell. -
Set up (8 hour) digestion.
26/08/2015: -
Analyse digestion. Showed a single band (a negative result). -
Put DH5αcells on to grow (for microscopy).
27/08/2015: -
First steps of slide preparation. -
Mini prepped Lpp OmpA full construct. -
Digested Lpp OmpA full construct with Age1. -
Ran an electrophoresis gel of the digestion - results were negative.
28/08/2015: -
Mini prepped Lpp OmpA full construct. -
Ran electrophoresis gel (of digestion with EcoR1+Nde1 and digestion with EcoR1+Age1). -
Rerunning electrophoresis gel (using old enzymes and Lpp OmpA plasmid full construct mini prep).
29/08/2015: -
Did gel extraction of Lpp OmpA full construct plasmid mini prep. -
No supernatant after freeze and squeeze step. -
MG1655Z1 cells transformed with PSB3K3 via electroporation.
30/08/2015: -
Redid gel extraction of Lpp OmpA full construct (plasmid mini prep) via.

Aug 24

Week 10

31/08/2015: -
Repeated gel extraction (not enough full construct mini prepped plasmid). -
Started mini prep of Lpp OmpA full construct transformed colony. -
Slide prep up to (but not including) Blocking Buffer wash. -
LB inoculated with colony from MG1655Z1 + pSB3K3 plate.
01/09/2015 -
Gel extracted anchor protein plasmids (from digestion). -
Microscopy session - some controls not the best, but significant difference in brightness of induced and Lpp OmpA full construct.
02/09/2015: -
PCR of sZFs 2, sZF10 sZFand 14. -
Mini prepped more plasmid from the working MG1655Z1cells. -
Set up more MG1655Z1cells to grow overnight. -
Restriction digestion of full construct plasmid carried out using Nhe1+Cla1.
03/09/2015: -
Triplicate mini prep made using colony 2 (MG1655Z1). -
Restriction digested mini prep. -
Ran a gel extraction of digestion, then extracted from gel. -
Ran a PCR of the cut zinc fingers.
04/09/2015: -
Mini prepped a large batch of Dh5α Z1cell. -
Ran a restriction digestion (using Age1+Nde1). -
JS006 transformed with PSB3K3 via electroporation, grown on 1:2000 Kanamycin plate. -
Successful PCR of sZF2 and sZF14 (purified and tested). -
Redid PCR of sZF10. -
Gel extraction of plasmid Mhe1+Cla1 digest, worked perfectly. -
PCR purified the 3 anchor proteins (BclA, pgsA and INP). -
Mini prepped anchor proteins. -
Digested the anchor proteins, ran on an electrophoresis gel and then extracted. -
Carried out a restriction digestion (with Age1+Nde1). -
Ran digestion on an electrophoresis gel, and then extracted.
05/09/2015 -
Gel extracted the plasmid digested by Nhe1+Cla1. -
Mini prepped more plasmid. -
Digestion of plasmid (using Nde1+Age1). -
Ran electrophoresis gel of Nde1+Age1 digestion.

Aug 31

Week 11

Sep 07

Final Week

Sep 14

@@ Line 63: / Line 63: @@
 Distribution Kit.
-<p><img src="https://static.igem.org/mediawiki/2015/0/01/Distribution_Kit_table_Lab_Book.png" height="120px" width="800px" border="1px"></p>
+<p><img src="https://static.igem.org/mediawiki/2015/0/01/Distribution_Kit_table_Lab_Book.png" height="60px" width="400px" border="1px"></p>
 <br>09/07/2015:
@@ Line 69: / Line 69: @@
 - <br>Nanodrop results for mini-prepped plasmids.
-<p><img src="https://static.igem.org/mediawiki/2015/7/7b/NanoDropResultsTableLab_BookiGEMWarwick.png" height="120px" width="800px" border="1px"></p>
+<p><img src="https://static.igem.org/mediawiki/2015/7/7b/NanoDropResultsTableLab_BookiGEMWarwick.png" height="60px" width="400px" border="1px"></p>
 <br>Ran an electrophoresis gel with plasmids from the mini-prep.