Team:Newcastle/Labwork/23 July 2009

=Formal Lab Session - 23rd July 2009=

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

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) BBa_C0056 -  2009 kit plate 1 – well 24A – plasmid pSB1A2
 * 2) BBa_B1002 – 2009 kit plate 1 – well 4B – plasmid pSB1AK3
 * 3) BBa_C0077 – 2009 kit plate 1 – well 14F – plasmid pSB2K3
 * 4) BBa_C0076 – 2009 kit plate 1 – well 14D – plasmid pSB2K3
 * 5) BBa_R0077 – 2009 kit plate 1 – well 10K – plasmid pSB1A2