Team:Cambridge/LabBook/Week11

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Week 11: Monday 20th September - Sunday 26th September

Contents

Monday

Tuesday

107. Expt: Biobrick assembly of pBAD+Luc (PP + LC) and (Emily)

  • Plasmids already extracted
  • Restriction digest following protocol on p88 using these quantities (in µl):
pBAD PP Luc (1) PP Luc (2) LC Luc (1)
Nuclease-free H20 14 14 14 14
10x FD Buffer 2 2 2 2
Plasmid DNA 2 2 2 2
FD EcoRI 1 1 1 1
FD SpeI 1 0 0 0
FD XbaI 0 1 1 1
  • Gel electrophoresis
    • pBAD failed - band at ~5000bp, should be 1200
    • others look about right ~ between 3 & 5kb (should be 3600bp)
  • Gel extraction - results in -20°C freezer

Wednesday

108. Expt: Send off PP + pSB1C3, EPIC pBAD for sequencing (Emily and Peter)

  • Miniprep EPIC pBAD and nanodrop
  • Prepare correct concentrations (100ng/µl for plasmid, 3.2pmol/µl for primers) to be sent off

109. Expt: Biobrick assembly of pBAD + PP Luc (1), PP Luc (2), LC Luc (1), PP+pSB1C3

  • Peter 'miniprepped' PP+pSB1C3 to extract plasmid
  • pBAD was amplified after trying to grow overnight culture failed. The following protocol was used:

Add to a PCR tube:

10µl 2x Phusion Master Mix
1µl VF2 Primer
1µl VR Primer
8µl HPLC H20
20µl
  • Use stab to extract colony from pBAD plate and swirl in tube. Run following program on PCR machine saved as 'pBAD Amplification':
    • Heated lid at 110°C
    • Denaturation for 1m30s at 98°C
    • Cycle 35 times
      • Denaturation for 30s at 98°C
      • Elongation for 2m at 72°C
    • Elongation for 7m30s at 72°C
  • PCR Purification of pBAD from PCR reaction was performed using Qiagen kit
  • Restriction: repeating restriction of LC Luc (1) because tube from yesterday was dropped. Use protocol on p88 with following quantities (in µl):
pBAD LC Luc (1)
HPLC H20 0 14
10x FD Buffer 2 2
Plasmid DNA 16 (PCR product)* 2
FD EcoRI 1 1
FD XbaI 0 1
FD SpeI 1 0

'*' Nanodrop said 29ng/µl after PCR Purification, which was strangely low. So used 16µl as recommended (in order to not exceed 0.2µg) but maximise amount of DNA.

  • Gel electrophoresis: Run gel with following quantities:
pBAD LC Luc (1)
DNA 17 17
6x Orange LD 3 3

110. Expt: Plate reading experiment to investigate effect of pH on light output

3pH: 5.3, 6.1, 7

D-luciferin and Caged D-luciferin (at 10mM mDMSO)

Plate layout:

pH 5.3 pH 6.1 pH 7.0 No buffer
D-luc o o o o o o o o o o o o
Caged D-luc o o o o o o o o o o o o
  • Arabinose: 100µM
  • Luciferin: 100µM
  • 3 wells with Arabinose only, and 3 blank wells. Buffer at 7.
  • 100µl per well
  • 50µl of cells
  • 48.5µl of Buffer
  • 1.5µl of Luc/1µl of caged Luc
  • 1µl of Arabinose

111. Expt: continuation of Biobrick assembly of pBAD with P.P.Luc(1), P.P.Luc(2), L.C.Luc(1), P.P.pSB1C3 (Emily)

  • Ligation: Accidentally forgot to restrict P.P.pSB1C3 so will do that later. Followed ligation protocol on p91 with following quantities:
P.P.Luc(1) P.P.Luc(2)
Vector DNA (µl) 7.8 8
pBAD (3:1 excess) (µl) 7.2 1
5x Rapid Ligation Buffer (µl) 4 4
T4 DNA Ligase (µl) 1 1
Nuclease-free H20 (µl) 0 4.6

Cells that were ligated were then transformed

There was not enough pBAD to ligate L.C.Luc(1) ==> PCR lots of pBAD.

pBAD Colony PCR

Mix in 3x PCR tubes:

Volume (µl)
Nuclease-free H20 20
2x Phusion MasterMix 25
VF2 Primer 2.5
VR Primer 2.5
pBAD stab stab from colony
50

Run program from p101.

  • PCR Purification: using Qiagen kit
  • Nanodrop:
    • Tube 1 --> 36.7ng/µl
    • Tube 2 --> 69.1ng/µl
    • Tube 3 --> 66.2ng/µl
  • Restriction: Following protocol on p88 with these quantities:
pBAD (1) PP Luc (2) PP pSB1C3 pBAD (2) pBAD (3)
Nuclease-free H20 0 14 13 1.6 1
10x FD Buffer 2 2 2 2 2
Plasmid DNA 16 2 3 14.4 15
EcoRI 1 1 1 1 1
SpeI 1 0 0 1 1
XbaI 0 1 1 0 0
  • Ran on a gel with 17µl DNA + 3µl of 6x orange LD in this order: Hyperladder I, pBAD 1, pBAD 2, pBAD 3, PP Luc2, PP pSB1C3
  • All bands were as expected: pBAD - 1200bp, PP Luc(2) - 3600bp, PP pSB1C3 - ~4500bp
  • Ben gel extracted

Ligation

Following protocol on p91, incubating at RT for 30 mins. The nanodrop reading for PPLuc(2) was so bad (2.8ng/µl) that the ligation was not performed because PPLuc(1) had already been transformed with a small amount of pBAD yesterday. Wait for results of that.

Nanodrops:

  • pBAD 1 -> 3.2ng/µl
  • pBAD 2 -> 34.5ng/µl
  • pBAD 3 -> 15.3ng/µl
  • PP pSB1C3 -> 11.5ng/µl
  • LC Luc(1) -> 53.2ng/µl
PP pSB1C3 LC Luc(1)
Vector DNA (µl) 11.9 5.9
pBAD 2 (3:1 excess) (µl) 3.1 9.1
5x Rapid Ligation Buffer (µl) 4 4
T4 DNA Ligase (µl) 1 1
Nuclease-free H20 0 0

Cells were then transformed.

Results

  • Glowing growth on PPLuc(2) plates and PP pSB1C3 plates
  • Growth but no glow on LC Luc(1) plates
  • No growth on PP Luc(1) plates

Results later that day:

  • LC Luc(1) now glowing :)

Thursday

112. Expt: Plate reader experiment. G28 and effect of D-luciferin on LC+LRE

This experiment was designed to measure the effect of Arabinose on G28 light output and D-luc level on LC/Red mutant. The layout was as follows (Blanks contain 30µl H20 + 70µl LB):

0µM 1µM 3µM 10µM
B ooo ooo ooo ooo
30µM 100µM 300µM 1mM
C ooo ooo ooo ooo
3mM 10mM Blank
D ooo ooo ooo
0µM 1µM 3µM 10µM
E ooo ooo ooo ooo
30µM 100µM 300µM 1mM
F ooo ooo ooo ooo
Cells, no luc, no Ara Blank 16:H20, 84:LB
G ooo ooo

µl°C