• Home
• Team
• Project
• Parts
• Modelling
• Sponsors
• Lab Book

Project

• Overview
• Choices Rationale
• Characterisation
• Safety
• Judging Comments

Sub-projects

• Metal Intake/Efflux
• Metal Sensing
• Sporulation Tuning
• Population Dynamics
• Stochastic Switch
• Metal Sequester
• Chassis
• Promoter Library

Wet Lab

• Lab Book
• Protocols

Meetings

• Meetings Calendar

Planning

• Timeline
• Ideas

Links

• Helping other teams
• Ethics
• T-Shirts
• iGEM Meetup
• Our City
• Fun and Games
• Sponsors
• Useful Links
• Contact Us
• Press Coverage

Contents 1 Lab Work - 23/07/09 1.1 Introduction 1.1.1 Practical Outline 1.1.2 Preparing cultures containing pMUTIN4 1.1.3 Preparing the chosen BioBricks

Lab Work - 23/07/09

Introduction

Today we will carry out one of the last tasks remaining from our practice lab sessions - precipitating the ethanol from our DNA samples so that gel electrophoresis can be carried out. This can be seen at this link - Introductory Lab Session (23/07/09).

Today also marks the beginning of the real lab work sessions in which the work will be directly involved in implementing our system. The first real task involves growing up E.coli cultures containing the plasmid backbone pMUTIN4, which will allow us in the near future to transfer our chosen BioBricks from the E.coli cells we are working with into Bacillus subtilis.

Another task which we have to complete is to prepare the BioBricks which are needed for our system and are found in the Spring Distributions. These include:

1. [http://partsregistry.org/Part:BBa_C0056 BBa_C0056] – cI repressor (lambda phage)
2. [http://partsregistry.org/Part:BBa_B1002 BBa_B1002] – Terminator sequence
3. [http://partsregistry.org/Part:BBa_C0077 BBa_C0077] – CinR activator
4. [http://partsregistry.org/Part:BBa_C0076 BBa_C0076] – Autoinducer synthetase
5. [http://partsregistry.org/Part:BBa_R0077 BBa_R0077] – Promoter (CinR and HSL regulated, RBS+)

Practical Outline

1. Precipitate the ethanol from our DNA plasmids (containing the RFP and GFP genes) and analyse them through DNA gel electrophoresis - this is Introductory Lab Session work
2. Grow up some DH5alpha E.coli cells containing the pMUTIN4 plasmid vector
3. Hydrate and prepare BioBricks present in Spring Distributions.

Preparing cultures containing pMUTIN4

For this exercise 2 x 500ml flasks were used. Into each flask 100ml of LB solution was added (under aseptic conditions) and also to each flask 0.5ml of ampicillin was added (ampicillin resistance is conferred by the pMUTIN4 plasmid). A sample was then taken from an agar plate containing E.coli possessing pMUTIN4 and was added to each of the two flasks. The 500ml flasks were then left in the 37ºC shaking incubator overnight.

Preparing the chosen BioBricks

There are five BioBricks which are likely to be used by our system. They are:

1. [http://partsregistry.org/Part:BBa_C0056 BBa_C0056] – cI repressor (lambda phage)
2. [http://partsregistry.org/Part:BBa_B1002 BBa_B1002] – Terminator sequence
3. [http://partsregistry.org/Part:BBa_C0077 BBa_C0077] – CinR activator
4. [http://partsregistry.org/Part:BBa_C0076 BBa_C0076] – Autoinducer synthetase
5. [http://partsregistry.org/Part:BBa_R0077 BBa_R0077] – Promoter (CinR and HSL regulated, RBS+)

Each well containing these BioBricks was firstly identified and marked with a pen (in the corners of the wells). Once we had confirmed that the correct wells had been identified, a pipette containing 15ml of distilled water was then used to pierce the film lids of the wells and hydrate the DNA within. After a few rounds of mixing the DNA inside the wells, the BioBricks were extracted and placed into Eppendorf tubes which were transferred to the -20ºC freezer for storage.

The locations of the BioBricks can be found here – all are contained within the iGEM spring distributions. The hyperlinks for each BioBrick below will direct you to the distribution information page:

1. [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_C0056 BBa_C0056] - 2009 kit plate 1 – well 24A – plasmid pSB1A2
2. [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_B1002 BBa_B1002] – 2009 kit plate 1 – well 4B – plasmid pSB1AK3
3. [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_C0077 BBa_C0077] – 2009 kit plate 1 – well 14F – plasmid pSB2K3
4. [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_C0076 BBa_C0076] – 2009 kit plate 1 – well 14D – plasmid pSB2K3
5. [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_R0077 BBa_R0077] – 2009 kit plate 1 – well 10K – plasmid pSB1A2