"
Team:Cambridge/LabBook/Week12
From 2010.igem.org
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 |
