Team:Cambridge/LabBook/Week12

From 2010.igem.org

(Difference between revisions)
(New page: <div align="center">Week 11: Monday 27th September - Sunday 3rd October</div> ==Monday== µl°C)
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<div align="center">Week 11: Monday 27th September - Sunday 3rd October</div>
<div align="center">Week 11: Monday 27th September - Sunday 3rd October</div>
==Monday==
==Monday==
 +
===114. Expt: Continuation of cultures for Mexico (Peter)===
 +
Sent to Mexico using Interparcel/DHL.
-
µl°C
+
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:
 +
{|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
 +
 
 +
*Restrict using protocol on p88, using following quantities:
 +
{|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.
 +
 
 +
===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:
 +
{|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)===
 +
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====
 +
{|class="wikitable"
 +
|-
 +
|
 +
|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:
 +
{|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
 +
*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):
 +
{|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
 +
|}

Revision as of 12:07, 7 October 2010

Week 11: Monday 27th September - Sunday 3rd October

Contents

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
Retrieved from "http://2010.igem.org/Team:Cambridge/LabBook/Week12"