Team:Cambridge/Notebook/Week8

From 2010.igem.org

Notebook: Week 8

Viewing project diary

View protocols and data

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

Monday

Anja repeated lux operon extraction with pJS-555 instead of pSK-555 in the hope of getting better results.

Tuesday

Sadly no light from the cells transformed with the extracted lux operon.

Wednesday

Theo attempted to test induction of pBAD in the BMG Labtech plate reader

Thursday

We came in in the morning and viewed the results from the plate reader experiment that had been running overnight. The bacterial lux results were disappointing. Our positive control was emitting (blue) light. (See photo on the right), but the other cells which had been incubated with arabinose in the hope of inducing them had not emitted light.

But the most interesting results came from the cells expressing firefly luciferase ([http://partsregistry.org/Part:BBa_I712019 BBa_I712019]) under a consitutive promoter / RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J13002 BBa_J13002]. These showed low light output for 13 hours and then a rapid increase in luminescence, to a level which was sustained until the evening. We left the cells in the reader overnight to continue reading.

Friday

We got results from sequencing of what we hoped to be our first Biobrick. It was not what we had hoped for, which was dissapointing. Additionally, Theo miniprepped a lux operon assembled using Gibson techniques and found it was the wrong size, after restriction enzyme cutting and running on a gel.

But on a positive note synthesis from DNA 2.0 arrived, and preliminary results using CHBT combined with D-cysteine showed significant light output implying that functional LRE should allow recycling of oxyluciferin.

Saturday

Sunday

Theo made a tool to allow export of BioBricks with annotations.

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

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.