Team:Cambridge/LabBook/Week8

From 2010.igem.org

Week 8: Monday 30th August - Sunday 5th September

Contents

Monday

Result (from Expt. 68):

No growth overnight, 2 growths out of 3 plates (+1 fungus) after weekend of growing. No glow.

69. Expt: Growing up of cell cultures of above experimental results & of results on p49 (Will)

Strains G1, j1 and G28wh were grown up in 5ml LB + 2µl Chloramphenicol, and on plates containing LB+Cm.

Left to grow at 30°C for 24h.

Results: Strains grew but did not glow after 48h.

70. Expt: Miniprep pSB1C3 from colonies (Anja)

Suspended a 'streak' of bacteria transformed with BBa_J04450 (registry part in pSB1C3) in 250µl buffer P1. Followed 'Bench protocol: Qiagen Spin Miniprep Kit Using a Microcentrifuge'.

Nanodrop 12.8ng/µl

71. Expt: Gibson assembly of pBAD, luxCD,AB,EG and pSB1C3 (Anja)

PCR:

Template Primer f. Primer r. Amplified Fragment
I0500 prefix.f.pBadstart luxCstart.r.pBADend pBAD (A)
pJS555 pBADend.f.luxCstart luxAstart.r.luxDend luxCD (B)
pJS555 luxDend.f.luxAstart luxEstart.r.luxBend luxAB (C)
pJS555 luxBend.f.luxEstart suffix.rev.luxGend luzEG (D)
BBa_J04450 luxGend.for.suffix pBADstart.r.prefix pSB1C3 (E)

BBa_J04450: once purified (E), once colony PCR (EC)

Followed protocol on p38 for PCR mixtures and programs.

Gel electrophoresis

Self-cast 1% agarose gel loaded as follows:

Easyladder II A A B B C C D D E E EC EC
10µl 17µl 17µl 17µl 17µl 17µl 17µl 17µl 17µl 17µl 17µl 17µl 17µl

Run at 120V.

No bands were visible. Not even in the marker lane. Since a gel was that had been sitting on the bench for a few days and SYBR Safe Dye is light sensitive it is assumed that the dye had become non-functional.

A 1% Agarose gel was loaded as follows:

Easyladder II A A B C D E? EC?
10µl 12µl 12µl 12µl 12µl 12µl 12µl 12µl PCR Rxn
3µl 3µl 3µl 3µl 3µl 3µl 3µl 6x LD
5µl 5µl 5µl 5µl 5µl 5µl 5µl Nuclease-free H20

These were all mixed and spun down prior to gel loading.

Bands of correct sizes were observed in all cases.

Gel Extraction

DNA was extracted from the gel following the "Bench protocol: MinElute Gel Extraction Microcentrifuge protocol".

Tuesday

72. Expt: Continued Gibson assembly of pBAD, luxCD,AB,EG and pSB1C3 (Anja)

Fresh Gibson 1.33x Master Mix was prepared following the protocol on p40.

A Gibson Assembly reaction was prepared according to the protocol on p40, but taking twice the volume of the Master Mix (ie 30µl) and each fragment (ie 2µl). The reaction was incubated for 1h at 50°C.

Transformation

TOP10 cells, red strain and black strain were transformed with 10µl of Gibson reaction following protocol on p13+14. Plated 150µl on LB agar plates with Chl. (Since we were short of LB+Agar+Chl plates, two old LB agar + 5µg/ml Chl plates from PJ were used). Incubated at 30°C overnight.

73. Expt: Prepare DNA sequencing material for BioScience (Theo and Anja)

Extracted plasmids from TOP10 cells were transformed with pBAD, luxCD,AB,EG in pSB1C3 following the 'Qiaprep Spin Miniprep Kit Using a Microcentrifuge' protocol.

Experiment was cancelled (to be repeated on 01/09/10)

Wednesday

74. Expt: Prepare DNA sequencing material for BioScience (Anja)

Extracted plasmids from TOP10 cells were transformed with pBAD, luxCD,AB,EG in pSB1C3 following the 'Qiaprep Spin Miniprep Kit Using a Microcentrifuge' protocol.

Nanodrop measurements: 88.7ng/µl

Primers are at 100µM = 100pmol/µl --> 30x dilution --> 3.2pmol/µl

BioScience requests DNA concentrations of 100ng/µl for plasmids (Volume: 6µl) and 3.2pmol/µl (Volume: 10µl) for primers.

  • Tube 1: 6µl of TetR repr. prom, rbs, P.P.luc in pSB1C3 (purified) at 88.7ng/µl
  • Tube 2: 10µl Forward primer of 3.2pmol/µl (made up from 0.33µl 100pmol/µl primer and 9.67µl nuclease-free H20)
  • Tube 3: 10µl Reverse primer of 3.2pmol/µl (made up from 0.33µl 100pmol/µl primer and 9.67µl nuclease-free H20)

75. Expt: Repeat of PCR of pBAD+luxCDABEG using modified reaction conditions (Will)

Added on ice:

2x Phusion Master Mix 25µl
Forward primers 0.25µl
Reverse primers 0.25µl
Template DNA 2µl
Distilled (nuclease-free) H20 22.5µl

notation as on p38

Fragment Template Reaction
110°C heated lid, 1m30s denaturation
Promoter I0500 purified (James Brown) 35 cycles
CD pJS555 30s 98°C, 30s 53°C, 1m45s 72°C
AB pJS555 7m30 elongation
EG pJS555
pSB1C3 Plasmid extract from BBa_J04450 strain Same as above only 71°C

In addition EG was run in touchdown PCR.

Heated lid 112°C
Denaturation 98°C 1m30
Touchdown 70°C to 45°C 45 cycles
Elongation 72°C 5min

PCR products were loaded into 1g/100ml agarose gel with 20µl/100ml SYBR SAFE strain.

Per well 25µl product + 5µl Gel loading dye

Wells: Hyperladder I, A, B, C, D, E, Dtouchdown, Hyperladder I

76. Night of 1 September - Plate Reader

In two parts:

(A)

Arabinose induction of G28 (lux operon under pBAD)

Rows A and B: all LB+Chl (100µl)

Column Inoculated with Arabinose Conc x val
1 G28 0 x1, 11
2 G28 1µM x2, 12
3 G28 10µM x3, 13
4 G28 100µM x4, 14
5 G28 1mM x5, 15
6 G28 100mM x6, 16
7 pSB1C3 0 x7, 17
8 Nothing - LB only 0 x8, 18
9 Slock10 0 x9, 19
10 Empty well x10, 20

Incubated at 30°C

Plate reader protocol:

  • Lum reads: gain 3200, time = 10s
  • OD reads at 100µl (595nm)
  • 10 mins between readings

Layout:

1 8 12
D x21 x28 x32
E x33 x40

Thursday

77. Expt: PCR of pBAD, luxCD,AB,EG and pSB1C3 (Will and Anja)

PCR mixtures were made up according to p38.

Notation as follows:

Amplified fragment Template used
A pBAD I0500
B luxCD pJS555
C luxAB pJS555
D luxEG pJS555
Dp luxEG previously gel extracted D
E pSB1C3 BBa_J04450
Ec pSB1C3 colony with BBa_J04450
Ep pSB1C3 previously gel ext. E

A,B and C were run at the following conditions:

110°C heated lid
Initial denaturation 98°C 1.30min
Denaturation 98°C 30s Start cycle (35x)
Annealing 53°C 30s
Elongation 72°C 1.45min End cycle
Final elongation 72°C 7.30min
Store at 10°C

D was run at:

110°C heated lid
Initial denaturation 98°C 1.30min
Denaturation 98°C 30s Start cycle (35x)
Annealing 59°C 30s
Elongation 72°C 1.45min End cycle
Final elongation 72°C 7.30min
Store at 10°C

Dp, E, Ec & Ep were run at:

110°C heated lid
Initial denaturation 98°C 1.30min
Denaturation 98°C 30s Start cycle (35x)
Annealing 64°C 30s
Elongation 72°C 1.45min End cycle
Final elongation 72°C 7.30min
Store at 10°C

Gel Electrophoresis

In self-cast 1% agarose gels (with 30µl SYBR safe dye per gel) ran: 25µl PCR reaction + 5µl 6x orange LD (mixed prior to loading).

Ladder: Hyperladder I

A,B,C,E,Dp were cut out for gel extraction. To improve the yield of fragment E, bands from both gels were cut out and pooled.

78. Expt: Firefly Luciferase/Luciferin overnight plate reader experiments (+CHBT test) (Will)

Plate reader: OD, 100ml, luminescence gain 3200, read length 10s, reads every 10 minutes, overnight (from 1st to 2nd Sept)

Plate layout (X21 to 40):

1 2 3 4 5 6 7 8 9 10 11 12
D X21 22 23 24 25 26 27 28 29 30 31 32
200µM 200µM 100µM 100µM 40µM 40µM 200µM 200µM 100µM 100µM 40µM 40µM
E 33 34 35 36 37 38 39 40
200µM 200µM 100µM 100µM 40µM 40µM * *

'*' = 50µl broth + 50µl PBS

All have 50µl cell in broth + 50µl substrate solution.

  • X22-26 CHBT substrate solution: 0.4mM (400µM) in 5% DMSO in PBS
  • X27-32 Luciferin substrate solution: 0.4mM in 5% DMSO in PBS

For 200µl:

    • 12.1µl luciferin stock
    • 10µl DMSO
    • 177.9µl PBS 1x
  • X33-38 Luciferin substrate solution: 0.4mM in PBS

Luciferin, sodium salt, molar mass = 302g/mol

Stock at 2mg/µl = 0.002g/ml

0.002g/302g/mol = 6.62x10^-6 mol/ml or 6.62x10^-3 Molar solution (6.62mM)

We want 0.4mM so 6.62/0.4 = 16.5x dilution

Results

CHBT: no light

79. Expt: Transforming reporters (Theo)

Theo transformed TOP10cc with two translational units:

  • BBa_I15017 EYFP translational unit: B0032 RBS - plate 122M
  • BBa_I15016 ECFP translational unit - plate 122K

He plated out and made overnight cultures.

80. Expt: Gel extraction of pBAD, luxCD,AB,EG and pSB1C3 (continued from p65) (Will)

Bands were cut out of the gels and DNA was extracted following the 'Bench protocol: MinElute Gel Extraction Microcentrifuge Protocol'. The extracted DNA was stored at -20°C overnight.

81. Testing colonies from transformation on 31.08.10 for presence of Gibson assembled plasmid (Anja)

Colonies had grown on all plates from the transformation on 31.01.10, however, only after 3 days of incubating at 30°C.

Are these colonies just a result of degraded chloramphenicol or are they containing the desired plasmid?

A 10mM dNTP mix was prepared: 10µl 100mM dATP, dGTP, dTTP & dCTP + 60µl nuclease-free H20.

Colony PCR

Make up a master mix for 10 PCR reactions

10X NH4 Reaction Buffer 50µl
50mM MgCl2 10µl
dNTPs (10mM) 10µl
Forward Primer 10µl
Reverse Primer 10µl
BioTaw Polymerase 7.5µl
nuclease-free H20 400µl
~500µl

Aimed to amplify luxEG region, used according to primers (see p38).

Also prepared 10µM solutions of the two primers (using these rather than the 100µM stocks to reduce danger of contamination).

The Master Mix was aliquated in 50µls, to which bacteria from one colony each were added (dip pipette tip 1st into colony then aliquated PCR mix, suck up PCR mix in pipette tip, pipette up and down a few times).

PCR program (saved as BIOTAQ COL PCR) on G-storm:

110°C heated lid
Initial denaturation 96°C 1:30min
Denaturation 96°C 10s Start cycle (25x)
Annealing 60°C 20s
Elongation 72°C 1:30min (45s/kb) End cycle
Final elongation 72°C 5min
Store at 10°C

Gel Electrophoresis

1% Agarose E-gel was used. Loading mixtures contained 3µl 6x Orange LD, 7µl nuclease-free H20, 10µl PCR reaction mix

There were lots of primer dimers => BEN WAS RIGHT!

There is no band in any of the colonies of a size of ~1.8kb (corresponding to luxEG) => colonies do NOT contain desired plasmid :-(

Friday

82. Expt: Gibson assembly and transformation of pBAD, luxCD,AB,EG in pSB1C3 (continued from gel extraction on p.67) (Anja)

Gibson assembly reaction

30µl Gibson 1.33x Master Mix
2µl A
2µl B
2µl C
2µl D
2µl E
40µl

Incubated for 1h at 50°C

Transformation

Transformed 3x50µl TOP10 with 10µl Gibson reaction each. Following the protocol on p13+14. Plated 150µl on LB agar plates with Cm and incubated overnight at 37°C.

Results: On the 04.09.10 two of three transformations were observed to yield colonies (ie colonies on two of three plates), most of plates which (not all!) were pink. ==> Need to identify what plasmid they contain.

83. Expt: Digestions of rbs Luc, pBAD Slock Lux (G28), rbs+EYFP, rbs+ECFP (Theo)

Theo miniprepped these:

DNA (ng/µl)
G28 76
Luc 32
C 4.8
Y 12.7

Digestion:

  • G28 - SP (SpeI, PstI) - 13µl DNA
  • Luc - SP (SpeI, PstI) - 17µl DNA
  • C - XP (XbaI, PstI) - 17µl DNA
  • Y - XP (XbaI, PstI) - 17µl DNA

Fast digested for 30 mins

Gel run with Hyperladder I. Interpretation:

  • G28 is actually religated by pSB1C3 (but minus RFP)
  • Luc failed (but why?)
  • too little DNA from EYFP, ECFP => predictable (not from colonies)

Saturday

84. Expt: Recovering P. phosphoreum from old cultures for making glycerol stocks (Peter)

Grew up overnight culture from the old Falcon 4 in the morning the culture was turbid but not glowing on photos.

Streaked out two of the old phosphoreum lawns to achieve single, glowing colonies.

  • 5/9/10 => no colonies visible
  • 6/9/10 => tiny colonies visible, but not glowing on photos

Sunday

85. Expt: Glycerol stocks of TOP10 with tetR repressed rbs+luc+pSB1C3 (Peter)

Liquid cultures had grown in 37°C revolving incubator for two days. Broth appeared overgrown and sedimenting => resuspended sediment and streaked out onto Chl plates (37°C), placed liquid cultures into fridge.

6/9/10 put 9 single colonies in plate reader with some D-luciferin, after 6 hours ODs were up to ~1.5 but no glowing was detectable.

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