Team:Cambridge/LabBook/Week9

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Week 9: Monday 6th September - Sunday 12th September

Contents

Monday

86. Expt: Extract CDABEG from pJS555

  • Using 2 primers
    • prefix start of O
    • suffix end of G
  • Hope that it will glow in its own right without luxR+I
  • Need to put under a new promoter

87. Expt: Gibson transformation (cont. from p.70)

3 colonies on one plate were not pink. These were streaked out on new Chl plated and put into liquid cultures along with 2 of the red colonies.

Next check for the right sized fragment with colony PCR and try to induce with arabinose.

88. Expt: Plate Reader G28

Well Arabinose conc.
A1 0 x1,9
A2 1µM
A3 5µM
A4 10µM
A5 100µM
A6 Blank
A7 pSB1C3
A8 Blank x8,16

Reads every 10 mins

89. Expt: Extracting YFP, CFP, Luc tetR, G28 via miniprep (Paul)

Nanodrop readings (ng/µl)
YFP 33.3, 55.4
CFP 17.9, 20.4
Luc tetR 33.7
G28 125.3, 96.9
G28? 17.9

90. Expt: Colony PCR to extract the thioesterase gene from E. coli K12 (Paul)

We used TOP10 cells, a substrain of DH10B, which is a substrain of K12.

We did a colony PCR to isolate the gene:

3 replicates with: 1 negative control:
2x Phusion Mastermix 10µl 10µl
Template DNA 1µl 0
Primer 1 1µl 1µl
Primer 2 1µl 1µl
Nuclease-free H20 7µl 8µl

The negative control was to check for primer/dimer and contamination.

PCR protocol:

  • Initial denaturation: 5min @ 98°C
  • Touchdown: 16 cycles
    • Denaturation: 10s @ 98°C
    • Annealing: 20s @ 65°C to 57°C
    • Elongation: 15s @ 72°C
  • 30 cycles
    • Denaturation: 10s @ 98°C
    • Annealing: 20s @ 50°C
    • Elongation: 15s @ 72°C
  • End and hold at 10°C
  • We then ran result on a gel with SyberSafe at 6x lodaing Dye and Hyperladder IV


Lane: 1 2 3 4 5
Ladder Tube 1 Tube 2 Tube 3 Negative Control

Failed: Something obtained in control...

Nothing in other lanes

We repeated without touchdown PCR:

  • Denaturation 5min @ 98°C
  • 35 Cycles
    • Denaturation: 12s @ 98°C
    • Annealing: 20s @ 58.5°C
    • Elongation: 15s @ 72°C
  • Hold at 10°C

Failed again: Product in negative control observed again...


Tuesday

91. Expt: Further testing

Column Arabiniose/µl Well names
Repeat 1 Repeat 2 Repeat 3
1 0 X1 X12 X23
2 0 X2 X13 X24
3 0 X3 X14 X25
4 5 X4 X15 X26
5 10 X5 X16 X27
6 50 X6 X17 X28
7 100 X7 X18 X29
8 1000 X8 X19 X30
9 10,000 X9 X20 X31
10 Blank X10 X21 X32
11 Blank X11 X22 X33

Inoculation: 1ml overnight added to 3ml, Amp + Cm

92. Expt: Restriction Enzyme Digests

DNA Enzymes used Subsequent Nanodrop readings/ng/µl
1. Linear Plasmid EP 5.6
2. Luc TetR ES 4.0
3. Luc TetR EP 2.8
4. RBS YFP XP 19.4
5. RBS CFP XP 2.9
6. G28 lux SP 6.1

93. Expt: Extracting DNA 2.0 from Registry

  • Remove filter from plastic bag and place on a sterile and clean surface.
  • Add 100µl of 10mM Tris-HCl, pH 7.5 directly to the center of the filter
  • Incubate at room temperature for 2minutes.
  • Puncture the bottom of at 0.6ml tube using a syringe.
  • Place filter in the 0.6ml tube and place 0.6ml tube in a 1.5ml tube.
  • Place the 1.5ml tube(now containing punctured 0.6ml tube with filter) in tabletop centrifuge.
  • Spin 1 minute at full speed. The DNA containing liquid will transfer from the filter in the 0.6ml tube into the 1.5ml tube.
  • Discard the 0.6ml tube with filter. The 1.5ml tube now contains ~90µl buffer+DNA.
  • Carefully remove supernatant. there may be a small pellet consisting of filter debris. This pellet does NOT contain any of the DNA. The supernatant should contain approximately 2µl plasmid DNA(~20ng/µl).

The isolated DNA can subsequently be transformed, cut with restriction enzymes, or sequenced without further purification.

Then transformed using standard protocol

Result: Colonies all grew

Wednesday

94. Expt: Ligating restriction digests from yesterday

Theo wished to prepare three constructs:

  • A: P[TetR repressed] - WT luciferase - YFP in pSB1C3
  • B: pBad Lux aoperon (G28) - YFP in pSB1C3
  • C: P[TetR repressed] - WT luciferase

Volumes all in µl

pSB1C3 linearised cut with EP. Luc cut with ES. Luc cut with EP. G28. YFP. Rapid ligation buffer. T4 ligase.
A 4 9 0 0 2 4 1
B 4 0 0 9 2 4 1
C 2 0 13 0 0 4 1

Theo then incubated for 5mins at 22°C and following transformation protocol, plating out on Chlor plates.

95. Expt: Colony PCR of cells transformed with the Gibson assembly product (pages 70,73) (Done by Peter)

Took 2 colonies of plates ABCDE each

ran PCR on G-storm ('Phusion Lng rng clny PCR')

27 cycles

  • melting: 98µl 15s
  • annealing: 60µl 10s
  • extension: 72µl 3min

final elongation 10min

Ran on E-gel:

faint band at ~2000bp

no band visible at the expected 6.6kbp

PCR tubes placed in Freezer, ~30µl left

96. Expt: Colony PCR to extract the thioesterase gene from E. coli K-12 (repeat using new protocol)

We used K-12 strains from Veio collection ( JW3582 and JW5313) but during the cell lysis protcols writing on tubes was erased so we couldn't identify the 2 strains.

Cell lysis

  • Heat at 98 for 10 min
  • Freeze at -80 for 10min
  • Vortex for 2min

Cells were collected from plates and put in 20 of dH20

We performed 2 PCR (normal and touchdown) with 2 replicates for each strain and one negative control:

negative control
7µl of dH20 8µl of dH20
10µl of 2x Phusion Mastermix 10µl of 2x Phusion Mastermix
1µl of primer 1 @0.5mM 1µl of primer 1 @0.5mM
1µl of primer 2 @0.5mM 1µl of primer 2 @0.5mM
1µl of Cell suspension

PCR

  • Denaturation: 5min @ 98°C
  • 35 cycles:
    • 12s @ 98°C : Denaturation
    • 20s @ 58°C : Annealing
    • 20s @ 72°C : Elongation
  • Elongation: 5min @ 72°C
  • Hold at 10

Touchdown PCR

  • 10 cycles
    • Denaturation: 12s @ 98°C
    • Annealing: 20s @ 65 to 55°C
    • Elongation: 20s @ 72°C
  • 35cycles
    • Denaturation: 12s @ 98°C
    • Annealing: 20s @ 55°C
    • Elongation: 20s @ 72°C
  • Elongation 5min @ 72°C

We then ran a gel

Products were obtained for strain 2 in both Touchdown lanes and in one normal PCR lane

Thursday

97. Expt: Restriction Digest of DNA2.0 stuff, tetR and pSB1C3 with RFP

Miniprepped:

  • DNA2.0 P. pyralis (x2)
  • DNA2.0 L. cruciata (x2)
  • TetR promotor
  • pSB1C3 + RFP

Nanodrop results:

On the side of the tubes in red pen

Restriction enzymes:

  • DNA2.0 P.P. --> E + P
  • DNA2.0 P.P. --> X + P
  • DNA2.0 L.C. --> E + P
  • DNA2.0 L.C. --> X + P
  • TetR --> E + S
  • pSB1C3 RFP --> E + P

Ran on gel, results in lab book. Look correct.

98. Expt: PCR to add 20bp to pSB1C3 to be able to Gibson it with Thioesterase from p83

PCR protocol similar to p83 but with:

  • Denaturation 30s @ 98°C
  • 35 cycles:
    • Denaturation 12s @ 98°C
    • Annealing 20s @ 70°C
    • Elongation 45s @ 72°C
  • Elongation 7:30min @ 72°C
  • Hold at 10°C

It failed.

2nd attempt with annealing @ 69°C

We obtained results which were then extracted from the gel by Ben. We also added pure plasmid to compare intensities.

  • Lane 1: 1.8ng/µl
  • Lane 2: 2.3ng/µl
  • Lane 3: 4.5ng/µl
  • Lane 4: 24.5ng/µl

99. Expt: Gibson assembly of fragments from p87 and p84 (pSB1C3 and Thioesterase)

Readings for thioesterase extracted from expt p84 were as follows:

  • Lane 4: 5.7ng/µl
  • Lane 8: 12.7ng/µl
  • Lane 9: 12.4ng/µl

We performed more assembling:

  • Plasmid 4 --> Thio8
  • Plasmid 4 --> Thio9
  • Plasmid 3 --> Thio4
  • Plasmid 4 --> Thio4
  • Plasmid 1 --> Thio8
  • Plasmid 1 --> Thio9
  • Plasmid 3 --> Thio8
  • Plasmid 3 --> Thio9
  • Plasmid 2 --> Thio8

We used the standard protocol (OpenWetWare). We then ran gel with Lane 10 pSB1C3 from the registry as control.

We extracted the DNA samples marked in red (as the resulting plasmid should be circular so gel speed could be variable).

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