Team:Cambridge/Notebook/Week12

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Notebook: Week 12

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Week 12: Monday 27th September- Sunday 3rd October

Monday

We had begun to collaborate with the UNAM-Genomics_Mexico team, they have catalogued our communications [http://openwetware.org/wiki/IGEM:UNAM-Genomics_Mexico/2009/Notebook/Collaborations.Cambridge/2010/09/18 in detail]. We had given them advice on protocols but they were not having any more success so at their request Peter sent them DNA for 5 constructs, luxCDABE and Luciferase and LREs from both Luciola cruciata and Photinus pyralis with and without promoters.

Paul's plate reader experiments continued to give somewhat unexpected results, with an increase in light emission which seemed to be correlated with entry into stationary phase. We wanted to know if this was some strange promoter effect or a feature of the luciferase pathway. For these purposes Emily and Bill attempted to assemble fluorescent proteins both under pBAD alone and under pBAD with the rest of the operon. Paul set up another plate reader experiment with a wide range of arabinose concentrations used for induction. Will performed further site-directed mutagenesis of LC luciferase, in case Ben's failed.

Tuesday

Emily and Bill continued trying to assembly the fluorescent proteins with pBAD and PP luciferase. The process proved more difficult than we had hoped, but we got there eventually.

Theo also continued experimenting with his electrical engineering skills with the E.glometer: trying to build a DIY light measurement device.

In the evening we made a live recording of the Gibson Assembly Song at Bill's so that we could record the music video in the lab tomorrow.

Wednesday

Paul continued with his plate reader experiments, this time comparing the light output from all the different L. cruciata luciferase colour mutants.

The transformations from the fluorescent proteins showed some very small colonies. We left them to grow for longer.

We had a lot of fun recording the Gibson Assembly Song video in the lab today. Theo has very good camera skills.

Thursday

Today there were decent sized colonies on the fluorescent protein plates. However, it looked like there was a lot of cross-contamination - red colonies on lots of different plates that shouldn't have RFP. Very confusing. After examining the plates we just took colonies that appeared to be excited at the correct wavelengths and glow the right colour.

Ben, Emily and Bill recorded the individual tracks for the Gibson Assembly Song which Bill mixed using Garage band. It's slowly coming together.

Friday

Aware that next week we would need to pack up the lab to move into Jim Ajioka's lab in parasitology next week, we started slowly tidying up the lab :( It was a sad time indeed. Especially when the motivational posters of Anja had to come down off the walls.

We also realised clearing up the lab might take a while.

Saturday

Sunday

Week 12: Monday 27th September - Sunday 3rd October

Monday

114. Expt: Continuation of cultures for Mexico (Peter)

Sent to Mexico using Interparcel/DHL.

Tracking number 903532038716

Paid for by Peter: £26.50

115. Expt: Biobrick assembly of fluorescent proteins (YFP, CFP, RFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily and Bill)

Miniprepped overnight cultures of YFP, CFP, RFP with rbs's and PP+pBAD+pSB1C3
Nanodrop readings:

	Nanodrop reading (ng/µl)
Cm+PP+pBAD+pSB1C3	48.5
YFP+rbs	74.8
RFP+rbs	32.9
CFP+rbs	46.7

pSB1C3 from freezer was used at 12.9ng/µl

Restrict using protocol on p88, using following quantities:

	Cm+PP+pSB1C3	YFP (for PP)	YFP (for pBAD)	RFP (for PP)	RFP (for pBAD)	CFP (for PP)	CFP (for pBAD)	pSB1C3
Nuclease-free H20	1	6	6	1	1	6	6	1
10x FD Buffer	2	2	2	2	2	2	2	2
Plasmid DNA	15	10	10	15	15	10	10	16
EcoRI	0	0	0	0	0	0	0	1
SpeI	0	1	0	1	0	1	0	0
XbaI	1	0	1	0	1	0	1	0
PstI	1	1	1	1	1	1	1	1
	*	*		*		*

We ran this gel on an E-gel and realised that those lanes with '*' had been cut with the wrong enzymes - PP+pSB1C3 should have been cut with S+P, the RFPs should have been cut with X+P.
We carried on with those that had worked with just pBAD.

See continuation below.

116. Expt: Plate reader readings for Arabinose induction for LCLuc+PPLuc under pBAD

Arabinose concentration varying from 0µM to 10mM

1. 0µM: 0Ara/30µl of H20
2. 1µM: 1µl of Ara@100µM/29µl of H20
3. 3µM: 3µl of Ara@100µM/27µl of H20
4. 10µM: 10µl of Ara@100µM/20µl of H20
5. 30µM: 30µl of Ara@100µM/no H20
6. 100µM: 1µl of Ara@10mM/29µl of H20
7. 300µM: 3µl of Ara@10mM/27µl of H20
8. 1mM: 10µl of Ara@10mM/20µl of H20
9. 3mM: 30µl of Ara@10mM/no H20
10. 10mM: 1µl of Ara@1M/29µl of H20
11. No luciferin, 100µM Ara => 1µl of 10mM, 30.5 of H20

D-luciferin at 100µM -> 15µl.

Total volume 100µl per well. 60.5µl of overnight culture

Plate layout:

	1-3	4-6	7-9	10-12
A	PP(1)	PP(2)	PP(3)	PP(4)
B	PP(5)	PP(6)	PP(7)	PP(8)
C	PP(9)	PP(10)	PP(11)	LC(5)
D
E
F	LC(1)	LC(6)	LC(3)	LC(4)
G			LC(7)	LC(8)
H	LC(9)	LC(10)	LC(11)	Blanks

117. Expt: Repeat of Ben's experiment of altering the other 4 sites of LC luciferase using directed mutagenesis (Will)

PCR mixes were:

0.25µl forward primer
0.25µl reverse primer
2µl template
22.5µl PCR water
25µl 2x Phusion Mastermix

Template DNA was taken from colony (100µl PCR water + 1 colony)
Primers were taken directly from tubes (undiluted)

PCR Protocol

	110°C	Heated lid
1m30	98°C	Denaturation
Cycle 30 times
30s	98°C	Denaturation
2m	72°C	Annealing & Elongation
End cycle
7m30	72°C	Final elongation
	10°C	Final hold

Did not work - abandon experiment

118. Continuation of Biobrick assembly of fluorescent proteins (YFP, RFP, CFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily)

Lengths of proteins cut with X+P were right
Gel extraction was performed using Qiagen protocol

Ligation

Followed protocol on p91 using following quantities:

	RFP	CFP	YFP
5x Rapid Ligation Buffer	4	4	4
T4 DNA Ligase	1	1	1
pSB1C3	5.7	5.4	6.2
DNA	8.1	8.5	7.5
pBAD (34.5ng/µl)	1.2	1.1	1.3

Cells were transformed on 28/9/10 overnight after leaving them to air under fume hood.

Tuesday

Restriction again, repeating from 27/9/10 using the correct restriction enzymes:

	Cm+PP+pSB1C3	YFP (for PP)	RFP (for PP)	CFP (for PP)
Nuclease-free H20	1	6	6	6
10x FD Buffer	2	2	2	2
Plasmid DNA	15	10	10	10
SpeI	1	0	0	0
XbaI	0	1	1	1
PstI	1	1	1	1

All showed correct bands on gel
Gel extraction was performed
Ligation was performed:

	YFP (4.8ng/µl)	RFP (12.1ng/µl)	CFP (9.5ng/µl)
5x Rapid Ligation Buffer	4	4	4
T4 DNA Ligase	1	1	1
DNA	4.7	2.3	2.8
PP+pSB1C3	10.3	12.7	12.2

Transformation into TOP10cc from freezer on Cm+Ara plates
Also transformed plambda+rbs from registry onto Cm plate. Should have been on Amp plate so did not grow.

Results

After 1 day - little colonies on all plates
After 2 days - looks like lots of cross-contamination. Only some colonies glow. Streaked out interesting colonies + grew up overnight culture

Wednesday

119. Expt: Plate reader of:

bacteria containing PP luciferase under pBAD at varying arabinose concentrations, with & without LRE as part of the operon
bacteria containing:
- LC wt
- LC 239
- LC 326
- LC 433
- LC 452
- EPIC
Arabinose concentrations were set up as on p109
D-luciferin at 10mM, 1µl were added
Cells were taken from liquid culture apart from ... which was taken from solid culture into LB

Layout (PP LRE = luciferase with LRE, PP = luciferase without LRE):

	1-3	4-6	7-9	10-12
A	PP LRE (1)	PP LRE (2)	PP LRE (3)	PP LRE (4)
B	PP LRE (5)	PP LRE (6)	PP LRE (7)	PP LRE (8)
C	PP LRE (9)	PP LRE (10)	PP LRE (11)
D	PP (1)	PP (2)	PP (3)	PP (4)
E	PP (5)	PP (6)	PP (7)	PP (8)
F	PP (9)	PP (10)	PP (11)
G	LC wt	LC 239	LC 326	LC 433
H	LC 452	EPIC		LB+water