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Meetings Calendar

Formal Lab Session - 17th August 2009

Team Newcastle iGEM 2009 17-08-09 IMG 0437.JPG

Overview

Metal Sensor Team - tested the amyE knockouts in the B. subtilis pGFP-rrnB transformants (plated on LB + Chloram + starch) using iodine

Stochastic Switch Team - hydrated 7 BioBricks from Spring Distribution and transformed E. coli with them. Also analysed B. subtilis transformants using iodine

Sporulation Tuning/Chassis Team - carried out an improved second attempt at recovering cw1D spores and also analysed B. subtilis transformants with iodine.

Metal Sensor Team

Metal Sensor Team: LB + starch plate containing Bacillus subtilis with disabled amyE after being treated with iodine - Note there are no halos around the bacteria

LB + starch plates containing transformed Bacillus subtilis (with amyE disabled) being treated with iodine crystals - plates prepared by both Metal Sensing team and Stochastic Switch team

Introduction and Summary

In our last lab session (14/08/09) starch agar plates were made and once this task had been completed, Bacillus subtilis was entered onto the plate. This was done by firstly drawing a grid of 46 squares on the base of the agar plate and then marking each square with the bacteria. Into square 1, untransformed wild type Bacillus subtilis was added and into squares 2-46, Bacillus subtilis transformed with gfp-rrnb was added. These plates were then grown overnight.

The reason for this exercise is to see whether the bacteria that have survived the chloramphenicol treatment really have been given this property by taking up gfp-rrnb. If the bacteria can't break down the starch which surrounds them on these plates then they have surely taken up the plasmid vector; if they can break down the starch then they might have received the resistance by other means. It may also mean that the vector may have integrated into another area in the Bacillus's chromosome. Today we shall be adding iodine to the plates so that we can assess whether the transformed B. subtilis can break down starch.

What we did

The overnight plate with the B. subtilis growing on it was removed from the fridge (where it had been stored) and taken to the fume cupboard. This agar plate (with the lid removed) was then placed directly over another plate containing iodine crystals (the lid of this plate was also removed). The surface of the agar plate (containing B. subtilis cultures) was directly facing the iodine crystals so that any iodine vapour would hit the colonies of bacteria and the starch surrounding them.

Observations and Results

Before the iodine process was carried out it was noted that no colonies grew in square 1. In this square wild type B. subtilis (which has the amyE gene intact) was plated. The reason for no growth was due to it's lack of resistance to chloramphenicol (an antibiotic added to the starch plate). However in the other squares, which contained 'transformed' Bacillus subtilis, there were colonies present as resistance had been inherited.

After a few seconds of exposure the starch present in the agar plate began to darken and eventually turn black in the presence of iodine vapour. After a minute or so, the whole plate was blackened with iodine vapour.

When looking at the colonies in squares 2-46 there were no clear halos surrounding them. They were all surrounded by the blackened starch with no clear areas. This meant that the starch had not been broken down around them and therefore the bacteria had lost their ability to break down starch.

Conclusion

Stochastic Switch team: LB + starch plate containing Bacillus subtilis with disabled amyE after being treated with iodine - Note there are no halos around the bacteria

After attempting to transform Bacillus subtilis with gfp-rrnb, it appears that the bacteria have successfully taken up the vector plasmid and integrated it into it's own genome (in the correct area, i.e. in the amyE gene). It had been previously shown that the transformants were resistant to chloramphenicol, a property inherited from gfp-rrnb, and today it has been shown that the transformants have lost their ability to break down starch with amylase (a characteristic inherited when the gfp-rrnb plasmid integrates into the B. subtilis's chromosome at the amyE gene).

Stochastic Switch Team

Jess plates out the 'transformed' E. coli cells onto plates under aseptic conditions - not realising that the wrong procedure was used for transformations

This week the stochastic switch team will be rehydrating and transforming bricks from the distribution needed for the very last part of the lab work. The bricks we will be using are fror swapping the tester promoters in our synthesised stochastic construct with inducible promoters that will be linked to the rest of our project's systems.

Summary

Today we rehydrated 7 biobricks for the distribution: R0062; R0079; C0161; C0179; J44000; C0178; C0062. Which code for: LuxR promoter; LasR promoter; LuxI; LasR; HixC; LasI; LuxR respectively.

We rehydrated the bricks and transformed E.coli (DH5alpha) cells however we used the wrong protocol and therefore needed to repeat the transformations on Tuesday 18th. We also tested our Bacillus integrations from last week. In a fume cupboard we exposed our starch plates to iodine vapour. No halos could be seen around the colonies, so it was confirmed that the GFP-rrnb had integrated into the bacillus chromosome at the amyE locus.

Sporulation Tuning/Chassis Team

Summary

Jane and the plates for the cwlD spores.

Today, we plan to repeat the experiment which we did on the Wednesday,12th of August, which is the recovery of the cwlD spores.

We plan to follow the protocol for Method A again, however, this time, keeping in mind to add 40ul of our stock lysozyme instead of just 4ul.

We will carry out the usual treatment of the spores with lysozyme, buffer solution and L-alanine. However, we will also attempt one extra "treatment", which is carrying out the same "treatment", but without the addition of lysozyme. What we are trying to achieve here, is how great an effect lysozyme has on the spores as can been seen in the results section below.

The drawn-on sections for the single colonies of the transformations.

Also, we intend to plate out our transformed Bacillus subtilis onto starch plates so that we can carry out the iodine test on it to see if it has truly transformed. The rational and steps to carrying out the iodine test has been explained above by the Metal Sensor Team. The only difference is that our team only plated out 21 colonies; Square 1 contained the wild type Bacillus subtilis, which Square 2 to 21 contained the Bacillus subtilis transformed with gfp-rrnb as can be seen in the results section below.

Results

Recovery of cwlD Spores

The results were taken the following day, and the experiment seem to have been a success, as seen in the following photos.

This time round, the results observed seem to be more reasonable as compared to Wednesday, 12th August, as the control plate was not overgrown with colonies. This is probably due to the fact that the spores were "treated" as per normal, just without the addition of lysozyme. When lysozyme is added, it would disrupt the cell wall, thus it was thought that it would be a critical factor, which has been proved via this experiment.

cwlD mutant plates, control and concentration = 1

Observing the colonies growing on the plates, the experiment seems to be a success as the plate with a concentration of 1 has the most number of colonies, and the plate with a concentration of 10^-1 has less colonies than the plate with a concentration of 1.

The same pattern is observed for the other plates. The lower concentration plates have less colonies growing on it, which can be seen in the following pictures.

cwlD mutant plates, concentration = 10^-1 and 10^-2.

cwlD mutant plates, concentration = 10^-3 and 10^-4.

cwlD mutant plate, concentration = 10^-5.

The number of colonies recorded for each plate is presented in the following table:

Results for cwlD spores
Concentration	No. of Colonies
1	141 x 8 = 1128
10^-1	83 + 70 + 52 =205
10^-2	9
10^-3	2
10^-4	0
10^-5	0

Iodine Test on Transformed Bacillus subtilis with gfp-rrnb

After plating out single colonies from the transformed plates and incubating them overnight, the starch test, which consists of placing the incubated single colonies over some iodine crystals was performed the next day, to confirm that the transformed B.subtilis can break down starch.

The single colonies before going through the iodine test.

From the picture below, it can be seen that two of the single colonies plated out has halos around it, suggesting that it has not transformed properly.