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Team:Cambridge/LabBook/Week8
From 2010.igem.org
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 |
