From 2009.igem.org

Meetings Calendar

Formal Lab Session - 26th August 2009

Team Newcastle 2009 iGEM 26-08-09 IMG 0536.JPG

Overview

Stochastic Switch Team - carried out the restriction digests on the mini-preps reattempted yesterday (except for BBa_C0178)

Metal Sensor Team - digested BBa_J33206 midi-preps and analysed them through DNA gel electrophoresis

Promoter Library Sub-project - digested pGFP-rrnB plasmid with EcoRI and NheI

Stochastic Switch Team

Summary

Jess and Goksel working on the restriction digests

Today Lab started at 2pm, so we had the afternoon to do restriction digests for the remaining biobricks(other than C0178). Out of 12 cultures, 11 of them were grown. We used the fast digest system, which takes 5 minutes in a 37 degree waterbath rather than 60 minutes. Below are the volumes required for a double digest using EcoRI and PstI:

6ul water
2ul 10X Fast digest buffer
1ul of fast digest EcoRI enzyme
1ul of fast digest PstI enzyme
10ul DNA (miniprepped)

Total volume: 20ul

These were left for 5-10 minutes at 37 degrees, then 9ul of the total volume was added to 1ul of loading buffer, and the samples were run on the gel we prepared yesterday.

Running the gel

Jess innoculating 3ml of LB with DH5-alpha E.coli cells

We loaded the samples in the following order. all he biobricks had pSB1A2 as the plasmid backbone which is 2079bp long.

Sample	Location	Length
LuxR promoter	2,3	55bp
LasR promoter	4,5	157bp
LuxI CDS	6,7	585bp
LasR CDS	8,9	723bp
LasI	10	609
HixC	11,12	26bp

The bands at the bottom of the gel reflects the lengths of the biobricks truly. However, in some wells there are three bands although we used two restriction enzymes and there should have been two bands for each. This might due to using the fast digest enzymes and not waiting long enough before running the gel. We had run the gel for LasI using normal enzymes and had seen only two bands before.

Today we also prepared some overnight cultures of DH5alpha cells (2X 3ml plain LB) for the Genomic DNA prep which will be carried out tomorrow.

Metal Sensor Team

Introduction

Mathew pipetting digested pSB1A2 plasmid containing the BioBrick BBa_J33206 into gel

Yesterday's lab session saw a few things happen: 1)We prepared the 'products' of a PCR reaction on the genome of Bacillus subtilis transformed with pGFP-rrnB and put it through gel electrophoresis, 2)The midi-prep samples stored in the freezer underwent ethanol precipitation and DNA concentration and 3) Agarose gel was made up for restriction enzyme digests (to be done today). Time ran out quickly and this prevented the team from performing the other tasks. We hope to complete the tasks in today's lab session.

Today, the Metal Sensing team intend to digest some samples of BBa_J33206 in pSB1A2 with enzymes EcoRI and PstI and run both them and an undigested sample on gel. The results of this experiment will determine our next moves.

Practical Outline

Jane helping the Metal Sensing Team with lab housekeeping during waiting times

The following list consists of the tasks the Metal Sensing team hope to achieve by the end of the day:

Carry out the restiction enzyme digests of the midi-prep plasmid DNA (pSB1A2 containing BioBrick BBa_J33206) and run them on the gel.
Carry out the genomic DNA preparations.

Procedure

Restriction enzyme digests of the midi-prep DNA

It was decided that four samples would be prepared from our midi-prep stock of the BBa_J33206 BioBrick in plasmid pSB1A2 DNA. The four samples include sample DNA on it's own (uncut plasmid), sample DNA + EcoRI, plasmid + PstI and finally plasmid + EcoRI + PstI (should yield the plasmid backbone and BioBrick insert).

The protocol we used was one suggested by FastDigest, the company from which our restriction enzymes came from.

The contents of the digest samples are as follows:

Contents of the four samples of *BBa_J33206* in *pSB1A2* plasmid
	pSB1A2 alone	pSB1A2 + EcoRI	pSB1A2 + PstI	pSB1A2 + EcoRI + PstI
DNA (ul)	10	10	10	10
Restriction Buffer (ul)	2	2	2	2
Restriction Enzyme(ul)	0	1	1	2
Distilled water(ul)	8	7	7	6

The benefit of using FastDigest enzymes is that they only take 5 minutes to digest the DNA samples! To be certain that the DNA samples are cut correctly, the time given under incubation for the enzymes to digest the DNA was 10 minutes. After this time, the digests and the undigested sample were run on the gel

DNA gel electrophoresis of digests

The digest samples were treated in the way the DNA gel electrophoresis protocol suggested - 9ul of digest DNA was taken from each of the four tubes and to each, 1ul of loading buffer (dye) was added. 10ul of the samples were then added to the wells (located in between the HindIII DNA ladder filled wells) in the following order:

Lane 1 = blank
Lane 2 = 8ul of HindIII DNA ladder
Lane 3 = 10ul of pSB1A2 alone
Lane 4 = 10ul of pSB1A2 + EcoRI
Lane 5 = 10ul of pSB1A2 + PstI
Lane 6 = 10ul of pSB1A2 + EcoRI + PstI
Lane 7 = 8ul of HindIII DNA ladder

The DNA gel electrophoresis was run at 90v and run for 30 minutes. The results and analysis of the gel can be seen below in the 'Results' section

Results

Team Newcastle iGEM 2009 Geldoc 2009-08-26 16hr 330min.jpg

It is quite clear from this photograph that the restriction digests did not work properly. Whilst it looks like the plasmid backbones are the right size (we are expecting the backbone to be 2079bp and on the gel it appears to be between 1904 and 3530bp), their appearance looks unconvincing. All four plasmid backbones appear to have been split into two parts; this should not happen at all. The samples treated with only one enzyme should be linearised not cut into two parts whereas in the sample treated with both enzymes, the backbone should be in one piece with the insert removed.

It has to be noted that the insert appears smaller than expected; according to the Parts Registry the BBa_J33206 BioBrick is 956bp whereas the gel photograph suggests it's roughly 564bp long.

The suggested reasons for this are:

Too much DNA was added to the wells - this can be seen by the heavy black blots on the photograph
The FastDigest enzymes EcoRI and PstI were given too little time to act.

In another attempt of this restriction enzyme digest, the Metal Sensor team should consider altering the amount of contents in the restriction enzyme digest mix and should also consider reviewing the length of incubation time allowed for the digests to happen as well as how much DNA to put into the wells.

Promoter Library Sub-Project

The aim of this sub-project is to create a library of sigA promoters which differ in strength and to characterise them.

The first step in this process is to digest the pGFP-rrnb plasmid with restriction enzymes EcoRI and NheI. The enzyme digests were prepared in 1 Eppendorf tube in the following way:

15ul of pGFP-rrnb plasmid DNA (from a midiprep)
3ul NheI (FastDigest)
3ul EcoRI (FastDigest)
4ul FastDigest buffer
15ul distilled water

This makes up a total volume of 40ul The mixture was made in such a way that the DNA, the distilled water and the FastDigest buffer was added first before the enzymes were added. The tube was given a spin pulse and placed in a water bath at 37C for 1 hour.

After this the mixture was placed in the -20C freezer for use tomorrow.

In the meantime, 0.8% agarose gel was prepared in the usual way and poured into the gel tray. The only difference between this gel and others previously poured is that some of the comb was taped so that the wells formed were larger. Once the gel had set, 1xTAE buffer was sprinkled onto the surface and the gel wrapped up in cling film. The gel was placed in the fridge also for use tomorrow.