Team:Cambridge/LabBook/Week12

From 2010.igem.org

Week 12: Monday 27th September - Sunday 3rd October

Contents

Monday

114. Expt: Continuation of cultures for Mexico (Peter)

Sent to Mexico using Interparcel/DHL.

Tracking number 903532038716

Paid for by Peter: £26.50

115. Expt: Biobrick assembly of fluorescent proteins (YFP, CFP, RFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily and Bill)

  • Miniprepped overnight cultures of YFP, CFP, RFP with rbs's and PP+pBAD+pSB1C3
  • Nanodrop readings:
Nanodrop reading (ng/µl)
Cm+PP+pBAD+pSB1C3 48.5
YFP+rbs 74.8
RFP+rbs 32.9
CFP+rbs 46.7

pSB1C3 from freezer was used at 12.9ng/µl

  • Restrict using protocol on p88, using following quantities:
Cm+PP+pSB1C3 YFP (for PP) YFP (for pBAD) RFP (for PP) RFP (for pBAD) CFP (for PP) CFP (for pBAD) pSB1C3
Nuclease-free H20 1 6 6 1 1 6 6 1
10x FD Buffer 2 2 2 2 2 2 2 2
Plasmid DNA 15 10 10 15 15 10 10 16
EcoRI 0 0 0 0 0 0 0 1
SpeI 0 1 0 1 0 1 0 0
XbaI 1 0 1 0 1 0 1 0
PstI 1 1 1 1 1 1 1 1
* * * *
  • We ran this gel on an E-gel and realised that those lanes with '*' had been cut with the wrong enzymes - PP+pSB1C3 should have been cut with S+P, the RFPs should have been cut with X+P.
  • We carried on with those that had worked with just pBAD.

See continuation below.

116. Expt: Plate reader readings for Arabinose induction for LCLuc+PPLuc under pBAD

Arabinose concentration varying from 0µM to 10mM

  • 1. 0µM: 0Ara/30µl of H20
  • 2. 1µM: 1µl of Ara@100µM/29µl of H20
  • 3. 3µM: 3µl of Ara@100µM/27µl of H20
  • 4. 10µM: 10µl of Ara@100µM/20µl of H20
  • 5. 30µM: 30µl of Ara@100µM/no H20
  • 6. 100µM: 1µl of Ara@10mM/29µl of H20
  • 7. 300µM: 3µl of Ara@10mM/27µl of H20
  • 8. 1mM: 10µl of Ara@10mM/20µl of H20
  • 9. 3mM: 30µl of Ara@10mM/no H20
  • 10. 10mM: 1µl of Ara@1M/29µl of H20
  • 11. No luciferin, 100µM Ara => 1µl of 10mM, 30.5 of H20

D-luciferin at 100µM -> 15µl.

Total volume 100µl per well. 60.5µl of overnight culture

Plate layout:

1-3 4-6 7-9 10-12
A PP(1) PP(2) PP(3) PP(4)
B PP(5) PP(6) PP(7) PP(8)
C PP(9) PP(10) PP(11) LC(5)
D
E
F LC(1) LC(6) LC(3) LC(4)
G LC(7) LC(8)
H LC(9) LC(10) LC(11) Blanks

117. Expt: Repeat of Ben's experiment of altering the other 4 sites of LC luciferase using directed mutagenesis (Will)

PCR mixes were:

  • 0.25µl forward primer
  • 0.25µl reverse primer
  • 2µl template
  • 22.5µl PCR water
  • 25µl 2x Phusion Mastermix
  • Template DNA was taken from colony (100µl PCR water + 1 colony)
  • Primers were taken directly from tubes (undiluted)

PCR Protocol

110°C Heated lid
1m30 98°C Denaturation
Cycle 30 times
30s 98°C Denaturation
2m 72°C Annealing & Elongation
End cycle
7m30 72°C Final elongation
10°C Final hold

Did not work - abandon experiment

118. Continuation of Biobrick assembly of fluorescent proteins (YFP, RFP, CFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily)

  • Lengths of proteins cut with X+P were right
  • Gel extraction was performed using Qiagen protocol

Ligation

Followed protocol on p91 using following quantities:

RFP CFP YFP
5x Rapid Ligation Buffer 4 4 4
T4 DNA Ligase 1 1 1
pSB1C3 5.7 5.4 6.2
DNA 8.1 8.5 7.5
pBAD (34.5ng/µl) 1.2 1.1 1.3
  • Cells were transformed on 28/9/10 overnight after leaving them to air under fume hood.

Tuesday

  • Restriction again, repeating from 27/9/10 using the correct restriction enzymes:
Cm+PP+pSB1C3 YFP (for PP) RFP (for PP) CFP (for PP)
Nuclease-free H20 1 6 6 6
10x FD Buffer 2 2 2 2
Plasmid DNA 15 10 10 10
SpeI 1 0 0 0
XbaI 0 1 1 1
PstI 1 1 1 1
  • All showed correct bands on gel
  • Gel extraction was performed
  • Ligation was performed:
YFP (4.8ng/µl) RFP (12.1ng/µl) CFP (9.5ng/µl)
5x Rapid Ligation Buffer 4 4 4
T4 DNA Ligase 1 1 1
DNA 4.7 2.3 2.8
PP+pSB1C3 10.3 12.7 12.2
  • Transformation into TOP10cc from freezer on Cm+Ara plates
  • Also transformed plambda+rbs from registry onto Cm plate. Should have been on Amp plate so did not grow.

Results

  • After 1 day - little colonies on all plates
  • After 2 days - looks like lots of cross-contamination. Only some colonies glow. Streaked out interesting colonies + grew up overnight culture

Wednesday

119. Expt: Plate reader of:

  • bacteria containing PP luciferase under pBAD at varying arabinose concentrations, with & without LRE as part of the operon
  • bacteria containing:
    • LC wt
    • LC 239
    • LC 326
    • LC 433
    • LC 452
    • EPIC
  • Arabinose concentrations were set up as on p109
  • D-luciferin at 10mM, 1µl were added
  • Cells were taken from liquid culture apart from ... which was taken from solid culture into LB
  • Layout (PP LRE = luciferase with LRE, PP = luciferase without LRE):
1-3 4-6 7-9 10-12
A PP LRE (1) PP LRE (2) PP LRE (3) PP LRE (4)
B PP LRE (5) PP LRE (6) PP LRE (7) PP LRE (8)
C PP LRE (9) PP LRE (10) PP LRE (11)
D PP (1) PP (2) PP (3) PP (4)
E PP (5) PP (6) PP (7) PP (8)
F PP (9) PP (10) PP (11)
G LC wt LC 239 LC 326 LC 433
H LC 452 EPIC LB+water