Team:Newcastle/Labwork/17 August 2009

=Formal Lab Session - 17th August 2009=

= 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.

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
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
====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.

Summary


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.



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.

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.



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.







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

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.



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.