Team:Cambridge/Notebook/12

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<div align="center">Week 11: Monday 27th September - Sunday 3rd October</div>
 
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==Monday==
 
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===114. Expt: Continuation of cultures for Mexico (Peter)===
 
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Sent to Mexico using Interparcel/DHL.
 
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Tracking number 903532038716
 
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Paid for by Peter: £26.50
 
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===115. Expt: Biobrick assembly of fluorescent proteins (YFP, CFP, RFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily and Bill)===
 
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*Miniprepped overnight cultures of YFP, CFP, RFP with rbs's and PP+pBAD+pSB1C3
 
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*Nanodrop readings:
 
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{|class="wikitable"
 
-
|-
 
-
|
 
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|Nanodrop reading (ng/µl)
 
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|-
 
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|Cm+PP+pBAD+pSB1C3
 
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|48.5
 
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|-
 
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|YFP+rbs
 
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|74.8
 
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|-
 
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|RFP+rbs
 
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|32.9
 
-
|-
 
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|CFP+rbs
 
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|46.7
 
-
|}
 
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pSB1C3 from freezer was used at 12.9ng/µl
 
-
 
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*Restrict using protocol on p88, using following quantities:
 
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{|class="wikitable"
 
-
|-
 
-
|
 
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|Cm+PP+pSB1C3
 
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|YFP (for PP)
 
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|YFP (for pBAD)
 
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|RFP (for PP)
 
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|RFP (for pBAD)
 
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|CFP (for PP)
 
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|CFP (for pBAD)
 
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|pSB1C3
 
-
|-
 
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|Nuclease-free H20
 
-
|1
 
-
|6
 
-
|6
 
-
|1
 
-
|1
 
-
|6
 
-
|6
 
-
|1
 
-
|-
 
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|10x FD Buffer
 
-
|2
 
-
|2
 
-
|2
 
-
|2
 
-
|2
 
-
|2
 
-
|2
 
-
|2
 
-
|-
 
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|Plasmid DNA
 
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|15
 
-
|10
 
-
|10
 
-
|15
 
-
|15
 
-
|10
 
-
|10
 
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|16
 
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|-
 
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|EcoRI
 
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|0
 
-
|0
 
-
|0
 
-
|0
 
-
|0
 
-
|0
 
-
|0
 
-
|1
 
-
|-
 
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|SpeI
 
-
|0
 
-
|1
 
-
|0
 
-
|1
 
-
|0
 
-
|1
 
-
|0
 
-
|0
 
-
|-
 
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|XbaI
 
-
|1
 
-
|0
 
-
|1
 
-
|0
 
-
|1
 
-
|0
 
-
|1
 
-
|0
 
-
|-
 
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|PstI
 
-
|1
 
-
|1
 
-
|1
 
-
|1
 
-
|1
 
-
|1
 
-
|1
 
-
|1
 
-
|-
 
-
|
 
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|*
 
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|*
 
-
|
 
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|*
 
-
|
 
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|*
 
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|
 
-
|
 
-
|}
 
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*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.
 
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*We carried on with those that had worked with just pBAD.
 
-
 
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See continuation below.
 
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===116. Expt: Plate reader readings for Arabinose induction for LCLuc+PPLuc under pBAD===
 
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Arabinose concentration varying from 0µM to 10mM
 
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*1. 0µM: 0Ara/30µl of H20
 
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*2. 1µM: 1µl of Ara@100µM/29µl of H20
 
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*3. 3µM: 3µl of Ara@100µM/27µl of H20
 
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*4. 10µM: 10µl of Ara@100µM/20µl of H20
 
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*5. 30µM: 30µl of Ara@100µM/no H20
 
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*6. 100µM: 1µl of Ara@10mM/29µl of H20
 
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*7. 300µM: 3µl of Ara@10mM/27µl of H20
 
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*8. 1mM: 10µl of Ara@10mM/20µl of H20
 
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*9. 3mM: 30µl of Ara@10mM/no H20
 
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*10. 10mM: 1µl of Ara@1M/29µl of H20
 
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*11. No luciferin, 100µM Ara => 1µl of 10mM, 30.5 of H20
 
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D-luciferin at 100µM -> 15µl.
 
-
 
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Total volume 100µl per well. 60.5µl of overnight culture
 
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Plate layout:
 
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{|class="wikitable"
 
-
|-
 
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|
 
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|1-3
 
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|4-6
 
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|7-9
 
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|10-12
 
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|-
 
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|A
 
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|PP(1)
 
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|PP(2)
 
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|PP(3)
 
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|PP(4)
 
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|-
 
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|B
 
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|PP(5)
 
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|PP(6)
 
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|PP(7)
 
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|PP(8)
 
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|-
 
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|C
 
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|PP(9)
 
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|PP(10)
 
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|PP(11)
 
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|LC(5)
 
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|-
 
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|D
 
-
|
 
-
|
 
-
|
 
-
|
 
-
|-
 
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|E
 
-
|
 
-
|
 
-
|
 
-
|
 
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|-
 
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|F
 
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|LC(1)
 
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|LC(6)
 
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|LC(3)
 
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|LC(4)
 
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|-
 
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|G
 
-
|
 
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|
 
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|LC(7)
 
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|LC(8)
 
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|-
 
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|H
 
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|LC(9)
 
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|LC(10)
 
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|LC(11)
 
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|Blanks
 
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|}
 
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===117. Expt: Repeat of Ben's experiment of altering the other 4 sites of LC luciferase using directed mutagenesis (Will)===
 
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PCR mixes were:
 
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*0.25µl forward primer
 
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*0.25µl reverse primer
 
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*2µl template
 
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*22.5µl PCR water
 
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*25µl 2x Phusion Mastermix
 
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*Template DNA was taken from colony (100µl PCR water + 1 colony)
 
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*Primers were taken directly from tubes (undiluted)
 
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====PCR Protocol====
 
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{|class="wikitable"
 
-
|-
 
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|
 
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|110°C
 
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|Heated lid
 
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|-
 
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|1m30
 
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|98°C
 
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|Denaturation
 
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|-
 
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|Cycle 30 times
 
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|
 
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|
 
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|-
 
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|30s
 
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|98°C
 
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|Denaturation
 
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|-
 
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|2m
 
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|72°C
 
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|Annealing & Elongation
 
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|-
 
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|End cycle
 
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|
 
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|
 
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|-
 
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|7m30
 
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|72°C
 
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|Final elongation
 
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|-
 
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|
 
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|10°C
 
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|Final hold
 
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|}
 
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Did not work - abandon experiment
 
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===118. Continuation of Biobrick assembly of fluorescent proteins (YFP, RFP, CFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily)===
 
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*Lengths of proteins cut with X+P were right
 
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*Gel extraction was performed using Qiagen protocol
 
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====Ligation====
 
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Followed protocol on p91 using following quantities:
 
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{|class="wikitable"
 
-
|-
 
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|
 
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|RFP
 
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|CFP
 
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|YFP
 
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|-
 
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|5x Rapid Ligation Buffer
 
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|4
 
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|4
 
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|4
 
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|-
 
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|T4 DNA Ligase
 
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|1
 
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|1
 
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|1
 
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|-
 
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|pSB1C3
 
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|5.7
 
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|5.4
 
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|6.2
 
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|-
 
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|DNA
 
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|8.1
 
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|8.5
 
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|7.5
 
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|-
 
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|pBAD (34.5ng/µl)
 
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|1.2
 
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|1.1
 
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|1.3
 
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|}
 
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*Cells were transformed on 28/9/10 overnight after leaving them to air under fume hood.
 
-
 
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==Tuesday==
 
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*Restriction again, repeating from 27/9/10 using the correct restriction enzymes:
 
-
{|class="wikitable"
 
-
|-
 
-
|
 
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|Cm+PP+pSB1C3
 
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|YFP (for PP)
 
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|RFP (for PP)
 
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|CFP (for PP)
 
-
|-
 
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|Nuclease-free H20
 
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|1
 
-
|6
 
-
|6
 
-
|6
 
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|-
 
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|10x FD Buffer
 
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|2
 
-
|2
 
-
|2
 
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|2
 
-
|-
 
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|Plasmid DNA
 
-
|15
 
-
|10
 
-
|10
 
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|10
 
-
|-
 
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|SpeI
 
-
|1
 
-
|0
 
-
|0
 
-
|0
 
-
|-
 
-
|XbaI
 
-
|0
 
-
|1
 
-
|1
 
-
|1
 
-
|-
 
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|PstI
 
-
|1
 
-
|1
 
-
|1
 
-
|1
 
-
|}
 
-
 
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*All showed correct bands on gel
 
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*Gel extraction was performed
 
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*Ligation was performed:
 
-
 
-
{|class="wikitable"
 
-
|-
 
-
|
 
-
|YFP (4.8ng/µl)
 
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|RFP (12.1ng/µl)
 
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|CFP (9.5ng/µl)
 
-
|-
 
-
|5x Rapid Ligation Buffer
 
-
|4
 
-
|4
 
-
|4
 
-
|-
 
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|T4 DNA Ligase
 
-
|1
 
-
|1
 
-
|1
 
-
|-
 
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|DNA
 
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|4.7
 
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|2.3
 
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|2.8
 
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|-
 
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|PP+pSB1C3
 
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|10.3
 
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|12.7
 
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|12.2
 
-
|}
 
-
 
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*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
 
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**LC 326
 
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**LC 433
 
-
**LC 452
 
-
**EPIC
 
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*Arabinose concentrations were set up as on p109
 
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*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