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Formal Lab Session - 17th August 2009

Metal Sensor 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

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.

Sporulation Tuning/ Chassis Team

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 is 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.

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.

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.

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 to confirm that the transformed B.subtilis can break down starch.

July M T W T F S S     [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/1_July_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/2_July_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_July_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/4_July_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/5_July_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_July_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/7_July_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/8_July_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/9_July_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/10_July_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/11_July_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/12_July_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/13_July_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/14_July_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/15_July_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/16_July_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/17_July_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/18_July_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_July_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_July_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/21_July_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_July_2009&action=edit 22] [http://2009.igem.org/Team:Newcastle/Labwork/23_July_2009 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_July_2009 24] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/25_July_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_July_2009&action=edit 26] [http://2009.igem.org/Team:Newcastle/Labwork/27_July_2009 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_July_2009 28] [http://2009.igem.org/Team:Newcastle/Labwork/29_July_2009 29] [http://2009.igem.org/Team:Newcastle/Labwork/30_July_2009 30] [http://2009.igem.org/Team:Newcastle/Labwork/31_July_2009 31]

August M T W T F S S           [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/1_August_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/2_August_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_August_2009&action=edit 3] [http://2009.igem.org/Team:Newcastle/Labwork/4_August_2009 4] [http://2009.igem.org/Team:Newcastle/Labwork/5_August_2009 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_August_2009&action=edit 6] [http://2009.igem.org/Team:Newcastle/Labwork/7_August_2009 7] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/8_August_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/9_August_2009&action=edit 9] [http://2009.igem.org/Team:Newcastle/Labwork/10_August_2009 10] [http://2009.igem.org/Team:Newcastle/Labwork/11_August_2009 11] [http://2009.igem.org/Team:Newcastle/Labwork/12_August_2009 12] [http://2009.igem.org/Team:Newcastle/Labwork/13_August_2009 13] [http://2009.igem.org/Team:Newcastle/Labwork/14_August_2009 14] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/15_August_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/16_August_2009&action=edit 16] [http://2009.igem.org/Team:Newcastle/Labwork/17_August_2009 17] [http://2009.igem.org/Team:Newcastle/Labwork/18_August_2009 18] [http://2009.igem.org/Team:Newcastle/Labwork/19_August_2009 19] [http://2009.igem.org/Team:Newcastle/Labwork/20_August_2009 20] [http://2009.igem.org/Team:Newcastle/Labwork/21_August_2009 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_August_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/23_August_2009&action=edit 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_August_2009 24] [http://2009.igem.org/Team:Newcastle/Labwork/25_August_2009 25] [http://2009.igem.org/Team:Newcastle/Labwork/26_August_2009 26] [http://2009.igem.org/Team:Newcastle/Labwork/27_August_2009 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_August_2009 28] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/29_August_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/30_August_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/31_August_2009&action=edit 31]

September M T W T F S S   [http://2009.igem.org/Team:Newcastle/Labwork/1_September_2009 1] [http://2009.igem.org/Team:Newcastle/Labwork/2_September_2009 2] [http://2009.igem.org/Team:Newcastle/Labwork/3_September_2009 3] [http://2009.igem.org/Team:Newcastle/Labwork/4_September_2009 4] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/5_September_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_September_2009&action=edit 6] [http://2009.igem.org/Team:Newcastle/Labwork/7_September_2009 7] [http://2009.igem.org/Team:Newcastle/Labwork/8_September_2009 8] [http://2009.igem.org/Team:Newcastle/Labwork/9_September_2009 9] [http://2009.igem.org/Team:Newcastle/Labwork/10_September_2009 10] [http://2009.igem.org/Team:Newcastle/Labwork/11_September_2009 11] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/12_September_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/13_September_2009&action=edit 13] [http://2009.igem.org/Team:Newcastle/Labwork/14_September_2009 14] [http://2009.igem.org/Team:Newcastle/Labwork/15_September_2009 15] [http://2009.igem.org/Team:Newcastle/Labwork/16_September_2009 16] [http://2009.igem.org/Team:Newcastle/Labwork/17_September_2009 17] [http://2009.igem.org/Team:Newcastle/Labwork/18_September_2009 18] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_September_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_September_2009&action=edit 20] [http://2009.igem.org/Team:Newcastle/Labwork/21_September_2009 21] [http://2009.igem.org/Team:Newcastle/Labwork/22_September_2009 22] [http://2009.igem.org/Team:Newcastle/Labwork/23_September_2009 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_September_2009 24] [http://2009.igem.org/Team:Newcastle/Labwork/25_September_2009 25] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_September_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/27_September_2009&action=edit 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_September_2009 28] [http://2009.igem.org/Team:Newcastle/Labwork/29_September_2009 29] [http://2009.igem.org/Team:Newcastle/Labwork/30_September_2009 30]