Team:Cambridge/Notebook/12

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<div align="center">Week 11: Monday 27th September - Sunday 3rd October</div>
+
<html>
-
==Monday==
+
<div align="center">Week 12: Monday 27th September- Sunday 3rd October</div>
-
===114. Expt: Continuation of cultures for Mexico (Peter)===
+
</html>
-
Sent to Mexico using Interparcel/DHL.
+
-
Tracking number 903532038716
+
{{Team:Cambridge/Templates/Day|Day=Monday}}
 +
{{:Team:Cambridge/Templates/rightpic|src=Cambridge-Mon12.jpg}}
 +
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.
-
Paid for by Peter: £26.50
+
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.
-
===115. Expt: Biobrick assembly of fluorescent proteins (YFP, CFP, RFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily and Bill)===
+
{{Team:Cambridge/Templates/Day|Day=Tuesday}}
-
*Miniprepped overnight cultures of YFP, CFP, RFP with rbs's and PP+pBAD+pSB1C3
+
{{:Team:Cambridge/Templates/rightpic|src=Cambridge-Tue12.jpg}}
-
*Nanodrop readings:
+
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.
-
{|class="wikitable"
+
-
|-
+
-
|
+
-
|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
+
Theo also continued experimenting with his electrical engineering skills with the E.glometer: trying to build a DIY light measurement device.
-
*Restrict using protocol on p88, using following quantities:
+
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.
-
{|class="wikitable"
+
-
|-
+
-
|
+
-
|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.
+
{{Team:Cambridge/Templates/Day|Day=Wednesday}}
 +
{{:Team:Cambridge/Templates/rightpic|src=Cambridge-Weds12.jpg}}
 +
Paul continued with his plate reader experiments, this time comparing the light output from all the different L. cruciata luciferase colour mutants.  
-
===116. Expt: Plate reader readings for Arabinose induction for LCLuc+PPLuc under pBAD===
+
The transformations from the fluorescent proteins showed some very small colonies. We left them to grow for longer.
-
Arabinose concentration varying from 0µM to 10mM
+
We had a lot of fun recording the Gibson Assembly Song video in the lab today. Theo has very good camera skills.
-
*1. 0µM: 0Ara/30µl of H20
+
{{Team:Cambridge/Templates/Day|Day=Thursday}}
-
*2. 1µM: 1µl of Ara@100µM/29µl of H20
+
{{:Team:Cambridge/Templates/rightpic|src=Cambridge-Thu12.jpg}}
-
*3. 3µM: 3µl of Ara@100µM/27µl of H20
+
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.
-
*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.
+
Ben, Emily and Bill recorded the individual tracks for the Gibson Assembly Song which Bill mixed using Garage band. It's slowly coming together.
-
Total volume 100µl per well. 60.5µl of overnight culture
 
-
Plate layout:
 
-
{|class="wikitable"
 
-
|-
 
-
|
 
-
|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)===
+
{{Team:Cambridge/Templates/Day|Day=Friday}}
-
PCR mixes were:
+
{{:Team:Cambridge/Templates/rightpic|src=Cambridge-Fri12.jpg}}
-
*0.25µl forward primer
+
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.  
-
*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)
+
We also realised clearing up the lab might take a while.
-
*Primers were taken directly from tubes (undiluted)
+
-
====PCR Protocol====
 
-
{|class="wikitable"
 
-
|-
 
-
|
 
-
|110°C
 
-
|Heated lid
 
-
|-
 
-
|1m30
 
-
|98°C
 
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|Denaturation
 
-
|-
 
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|Cycle 30 times
 
-
|
 
-
|
 
-
|-
 
-
|30s
 
-
|98°C
 
-
|Denaturation
 
-
|-
 
-
|2m
 
-
|72°C
 
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|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:
 
-
{|class="wikitable"
 
-
|-
 
-
|
 
-
|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:
 
-
{|class="wikitable"
 
-
|-
 
-
|
 
-
|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:
 
-
{|class="wikitable"
 
-
|-
 
-
|
 
-
|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
+
==Saturday==
-
*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==
+
==Sunday==
-
===119. Expt: Plate reader of:===
+
<html>
-
*bacteria containing PP luciferase under pBAD at varying arabinose concentrations, with & without LRE as part of the operon
+
</div>
-
*bacteria containing:
+
</html>
-
**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):
+
-
{|class="wikitable"
+
-
|-
+
-
|
+
-
|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
+
-
|}
+

Latest revision as of 15:40, 27 October 2010

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

Contents

Monday

Cambridge-Mon12.jpg

We had begun to collaborate with the UNAM-Genomics_Mexico team, they have catalogued our communications 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

Cambridge-Tue12.jpg

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

Cambridge-Weds12.jpg

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

Cambridge-Thu12.jpg

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

Cambridge-Fri12.jpg

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