Team:Cambridge/Templates/HybridBook

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Revision as of 23:30, 9 August 2010

Notebook: Week 1

Project diary

Lab book

Week 1: Monday 12th - Sunday 18th July

Monday

{{:Team:Cambridge/Templates/{{{src}}}|src=Cambridge-Postit.jpg}} We spent the morning compiling a large set of ideas, each on its own post-it-note. We categorised them and excluded those that were clearly unfeasible. If you like, you can see the long list.

In the morning we narrowed them down to a shortlist of projects.

Tuesday

We spent the morning researching the shortlisted projects and seeing a demonstration of the Techne PCR machine by Bibby Scientific.

In the afternoon we worked on a practical with Qiagen equipment, helped by Simon Morgan. We miniprepped DNA, and then ran it on a gel.

Wednesday

In the morning we looked at past iGEM projects and compiled characteristics of successful projects, this was a very useful exercise.

We also presented the topics we had researched in detail to each other which gave us a better sense of which areas we wanted to pursue further.

After lunch we selected 4 projects to further work on. We swapped projects so that we could research a new topic and get up to speed on it, and also provide fresh insights.

We created pages for each project idea:

Thursday

We discussed quiescence in the morning, in particular the possibility of IP issues. We concluded we should consult David Summers who holds patents in the area. We decided there was not enough for a full project in the topic. We also considered whether altering RNA structure was a realistic aim for iGEM. Bio-Luminescence and Quiescence joint project -

A functional breakdown of the project was proposed:

Module 1 - Production of Luciferin, Luciferase and Luciferin Recovery enzyme (LRE) - these are all firefly
Module 2 - Biosynthetic pathway for luciferin
Module 3 - Biobricking quiescence - require a very good control system to prevent false activation, could add a conformational change to the molecule.

We discussed finding a "super" luciferase with 12x the affinity for the substrate and not for the product. The firefly systems are all eukaryotic, so we suggested that we could perhaps do this in yeast. Control of quiescence could also occur via cyclin control in yeast.

Friday

In the morning we decided to drop HIV project, due to the lack of easy signalling peptides. We continued research on Bioluminescence idea. We also set up a Flickr account.

Bill and Will attempted to estimate the luminosity of a bacterial colony, and compare this to the lower threshold of the human eye during scotopic vision. They used an estimate for the eye sensitivity using [1].

Theo predicted the structure of Rcd using mFold, but got a different conformation to the one shown in the paper, perhaps the algorithms used on the program have changed between now and when the paper was written.

We listened to Fireflies by Owl City and concluded the lyrics make no sense.

Saturday

Peter made a physical model of the X RNA to visualise the structure

Sunday

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)
12:20pm	-0.026	-0.032
2:30pm	0.437	0.530

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)
10:50am	0.008	-0.002
11:30am	0.013	0.000
12:00pm	0.023	0.024
12:35pm	0.032	0.034
01:00pm	0.040	0.046
01:32pm	0.052	0.063
02:00pm	0.073	0.090
02:35pm	0.106	0.140
03:00pm	0.137	0.174
03:25pm	0.179	0.230
03:38pm	0.203	0.256
03:55pm	0.225	0.313

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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