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Week 3: Monday 26th July - Sunday 1st August
Monday
Today we had another meeting in the morning to try and distribute jobs. Theo created a plan for an LRE Biobrick and submitted it to DNA 2.0 for a quote - we really wanted to get our DNA synthesised as quickly as possible.
Anja, Hannah and Emily had fun writing a draft article for [http://www.sterilin.co.uk/ Sterilin] describing what iGEM is, what we were hoping to accomplish and what Sterilin products we had been using. Peter and Will also created primer sequences for our first experiment. Quite a productive day!
Tuesday
Duncan gave us several E. coli strains today, so we started doing real, practical lab work! We plated out TOP10 to create colonies from which to create competent cells for transformation.
Duncan also brought us a lot of boxes of iGEM stocks from previous years. Emily, Peter, Theo and Anja spent the afternoon sorting through it and making towers of boxes in the lab - putting our tower building practice into use.
We also met with Dr. Summers to discuss quiescence. He was very encouraging but we have yet to settle IP concerns. He also suggested considering mopping up Rcd from a leaky inducible promoter with antisense RNA.
Wednesday
Today we inoculated a broth with TOP10. We are hoping to start making competent cells tomorrow!
Anja and Emily started planning the protocol for making the competent cells tomorrow.
Thursday
To do: prepare competent cells
Friday
- Preparing order for sythesis
- Theo aligned 300 luciferase-sequences to visualise consensus
- Peter and Theo discovered DINAmelt, downloaded UNA Fold to model Rcd dynamics
- Anja, Emily and Bill prepared chemically competent cells!
- By spending an hour in a cold room. It was cold but worth it. We got to drink hot chocolate.
Saturday
- Fernan tested our competent cells for us and they worked!
Sunday
- Anja, Bill, Emily and Theo discussed a final template for the wiki - it is looking pretty.
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Tuesday
1. Experiment: Streaking out of bacterial cultures (Peter & Anja)
On LB agar plates:
- TOP10
- MG1655
- W3110 hns 93-1
- BW25113
On LB agar + kan plates:
- BW25113 Δhns::kan
- BW25113 ΔtraA::kan
Incubated at 37°C overnight
Wednesday
all strains grew with individual colonies.
2. Experiment: Set up overnight culture of TOP10 (Emily & Anja)
Inoculated single bacterial colony (ATP10) in 12ml LB, incubated at 37°C with shaking (180 rpm) overnight.
Thursday
Inoculated 400ml LB with 5ml TOP10 overnight culture, incubate at 37°C with shaking (180rpm) put on at 11:40am
OD600 measurements:
| Time
| OD600 (1)
| OD600 (2)
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| 12:20pm
| -0.026
| -0.032
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| 2:30pm
| 0.437
| 0.530
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3. Preparation: CCMB80 Buffer (Volume500ml)
Arrows label dilutions to the indicated concentration
- KOAC 1M ---> 10mM 5ml
- CaCl2.2H20 1M ---> 80mM 40ml
- MnCl2.4H20 1M ---> 20mM 10ml
- MnCl2.6H20 1M ---> 10mM 5ml
- glycerol --->10%
- sterile H20 390ml
- prepare in flow hood
- sterile filter
- store at 4°C
4. Experiment: Set up overnight cultures of TOP10 (Will & Anja)
Inoculated single bacterial (TOP10) colony in 5ml SOB, incubated at rtp with shaking overnight
Friday
5.Experiment: Preparing chemicall competent cells (Emily, Bill & Anja)
(followed protocol for 'TOP10 chemically competent cells' from OpenWetWare)
Inoculated 800ml SOB with 3ml of TOP10 overnight culture (grown from single colony), incubated at 37°C with shaking (180rpm)
Put on at 9.45am
OD600 measurements:
| Time
| OD600 (teaching lab)
| OD600 (Jim's lab)
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| 10:50am
| 0.008
| -0.002
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| 11:30am
| 0.013
| 0.000
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| 12:00pm
| 0.023
| 0.024
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| 12:35pm
| 0.032
| 0.034
|
| 01:00pm
| 0.040
| 0.046
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| 01:32pm
| 0.052
| 0.063
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| 02:00pm
| 0.073
| 0.090
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| 02:35pm
| 0.106
| 0.140
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| 03:00pm
| 0.137
| 0.174
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| 03:25pm
| 0.179
| 0.230
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| 03:38pm
| 0.203
| 0.256
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| 03:55pm
| 0.225
| 0.313
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Cooled cells and CCMB80 buffer in ice bath for around 20min. Aliquoted 600ml culture in 12x50ml centrifuge tubes (pointed bottom). Centrifuged at 3000g at 4°C for 10min. Discarded supernatatent. Gently resuspended cell pellet in 20ml ice cold CCMB80 buffer per tube. Incubated on ice for 20min. Centrifuget at 3000g at 4°C for 10min.
Pooled Pairs of tubes together. 12 tubes ---> 6tubes.
Discarded supernatant. Resuspended pellet in 5ml ice for 20min. Aliquoted 150x200μl into Eppendorf tubes. Stored at -80°C.
Saturday & Sunday
6. Experiment: Measuring competency of TOP10 (Fernan)
TOP10 coopmetent cells (cc) taken out of -80°C freezer and put on ice. Check for thawing ater 5minutes (leave on ice).
Cut 1ml pipette tips with scissors sterilised in ethanol and flamed with a Bunsen burner.
Transfer ~50μl of TOP10 cc into 1.5ml Eppendorf tube with cut pipette tip. (will have to judge ~50μl by eye). Added 1μl of pUC19 (standard control plasmid) at 10-5μg/μl. Held on ice for 30min. Heat shockede for 60s at 42°C (waterbath). Put on ice for ~2min. Added 250μl pre-warmed (in 37°C incubator) SOC. Incubated at 37°C for 1h with rotation (for this purpose stick Eppendorf tubes in 12ml falcon tubes and put tape over the top). Plated 20μl on LB agar paltes with Amp (with blue shaped spreader). Grow colonies overnight at 37°C.
- Yielded ~150 cells/plate
- transformation efficiency (no. of transformed cells (colonies) generated by 1μg of plasmid DNA)
- volume of cells * colonies on a plate / mass of DNA transformed
- 15 * 150 * 105 = 2.25x108/ μgDNA
- volume=50μl cells + 1μl plasmid + 250μl SOC, 301μl of which 2Oμl were plated, giving dilution factor of 15
- mass of DNA is 10-5 μg
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