Team:Groningen/Template/MODULE/Notebook/Bandage

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Notebook > The Bandage

June 30 - July 4

Freeze drying L. lactis strain NZ9800 and NZ9700

These cells were stored at -80 °C

July 7 - July 11

Growing an over night culture of the freeze dried

The cells grew on the plate, meaning that L. lactis is able to survive the freeze drying

July 21- July 25

Growing a new overnight cultures and preparing the chemicals for the first polyacrylamide gels with L. lactis cells

Pouring a polyacrylamide hydrogel with Lactococcus lactis NZ9700 in it

Four polyacrylamide gels with different concentration were poured, one of 10 %, 15 %, 20 %, 25 %, and 30 % acrylamide, we decided to continue with 20 % for now

Pouring a polyacrylamide gel containing an overnight culture, this gel is divided in four, two gels were freeze dried, and two more were incubated overnight at 30 °C with fresh M17 medium

These gels were checked by phase contrast microscopy after incubation overnight, a lot of cells were visible, but we could not distinguish the living of dead bacteria, therefore we decided to continue the experiment with an inducable GFP expressing strain

July 28 - August 1

Preparing a GFP stock of L. lactis NZ9000 with pNZ8048g, and growing an overnight culture of this strain

Inducing the L. lactis NZ9000 with pNZ8048g with different concentrations of ZnSO4, the cells were observed under the microscope and some GFP expression was observed

Figure 1: The L. lactis NZ9000 strain with pNZ8048g under the microscope, on the left the GFP channel is shown, and on the right the ordinary phase contrast picture is displayed

4 August - 8 August

This time we'll try pouring a gel on ice. This is because during the solidifying of acrylamid there is a lot of heat, we were a bit scared this heat could destroy our cells. Unfortunately the solidifying process will slow down to a point that it will take more then an hour to complete. We've decided that this wouldn't work, so we continue without using ice.

Poured a gell with the GFP strain. Freeze a half, and the other half was put into the stove. The next day these were both incubated with ZnSO4 for an hour. After an hour of incubating this didn't give any results. Incubation time should be longer the next time.

Figure 2: The L. lactis NZ9000 strain with pNZ8048g under the microscope, on the left the GFP channel is shown, and on the right the phase contrast picture is displayed

August 11 - August 15

A polyacrylamide gel with the L. lactis NZ9000 strain with pNZ8048g, we incubated the gels in GM17 media with ZnSO4 for two hours at 30 °C, and observed the gels under the microscope

Some GFP expressing L. lactis was seen under the microscope

Repeating the experiment looking for the best conditions

Figure 3: The L. lactis NZ9000 with pNZ8048g, seen through a phase contrast microscope, on the right the GFP channel is displayed

August 18 - August 22

Pouring a polyacrylamide gel with an overnight culture of L. lactis NZ9000 with pNZ8048g, the gel was split into several parts, and the parts were observed after certain time-spams divided over two weeks

L. lactis NZ9000 with pNZ8048g seems to be able to survive the polyacrylamide is liquid state and the polymerization process

September 1 - September 4

Nisin diffusion tests performed with the polyacrylamide gel

A GM17 agar plate with L. lactis NZ9000 on it, this is a L. lactis strain that is sensitive for nisin, after which we made three holes in the agar how will we do this: We will start by pouring gm17 agar plates with the nz9000 strain on it, this is a strain that doesn't have any nisin resistance. While pouring we had 3 empty circles inside this agar plate where we can put in 3 small samples of acrylamid gel. This acrylamid gel contains the NZ9700 strain, this is a strain that can create nisin. It does this after induction with nisin.

Inside the three holes, polyacrylamide gels with the L. lactis nisin producing strain NZ9700

The three holes had slightly different conditions, one of the three wholes if filled with a gel, which was incubated overnight at 30 °C, the other two were filled with a freshly prepared gel of which one is not induced to produce nisin Our first try didn't go to well because the gel didn't seem to touch the agar, diffusion wouldn't start.

During the first experiment we hardly received any results, most likely this was caused by the fact that the gel did not touch the agar

Figure 4: The result of one of the diffusion tests, the halos around the gels shows the range of inhibition

September 8 - September 12

Performed the difussion experiment again, but using better conditions, the plate was incubated overnight at 30 °C

Figure 4 shows the result of a diffusion experiment under improved conditions, the inhibition area around the gel patches indicate the diffusion of nisin through the polyacrylamide gell

One of the more challenging parts of this project. Creating a carrier for our bacteria which is safe enough for normal usage.

Figure 5: An other diffusion experiment with the nisin producing L. lactis strain through acrylamid.

September 15 - September 19

Some more diffusion experiments were performed, this time the gels were washed before putting them in the agar plate with the nisin sensitive L. lactis strain

September 29 - October 3

During the last few week several time lapse experiments were performed, with all kind of concentration of polyacrylamide gel, and also with agar and agarose gells, in order to L. lactis grow in the gel

Small gels were poured and incubated overnight in GM17 at 30 °C, two types of gels were used, an intact one, and a fractured one, this might have some effect on releasing some L. lactis cells from the gel

Several percentages of polyacrylamide gels were made, from 2.5 % until 15 % (2.5 - 5 - 7.5 - 10 - 12.5 - 15), the intact gels showed no growth, therefore we can conclude that L lactis is not able to grow in any percentage of polyacrylamide gel

From 2.5 % until 15 % L. lactis was able to grow on/in the fractured polyacrylamide gel, no growth was show on the higher percentages

October 6 - October 10

New L. lactis NZ9000 with pNZ8048g were grew overnight in Chemically Defined Medium (CDM), and freeze dried the next day for several OD₆₀₀ values

One of the freeze dried samples was resuspended in 1 mL demi water to check for viability

The resuspended cells were inoculated in CDM in a 1:100 ratio and grown overnight at 200 rpm and 30 C

This week new cultures were grown for freeze drying. This was done to test the growth in chemically defined medium (CDM), which is the medium that will be used in the final bandage. Cultures with several OD₆₀₀ values were freeze dried in 5 ml samples. Unfortunately the freeze drying machine could only be used on the 9^th of October. Samples were taken from the machine on the 10^th of October and one of the samples was resuspended in 1 ml demi water to check for viability. The resuspended cells were inoculated in CDM in a 1:100 ratio and grown overnight at 200 rpm and 30 °C

October 13 - 19 October

The freeze dried cells, which were made previous week were used to prepare several gels for microscopy, the pore size of a polyacrylamide gel with a percentage of 2.5 % is approximately 200 nm and the pore size of a 10,5% gel is approximately 20 nm¹

The pore size of 1.5% low melting point (LMP)agarose is around 150 nm² as can be seen in figure 6. A L. lactis cell varies between sizes of 500nm up to 1500nm³

Figure 6: Pore sizes of LMP agarose gels in relation to the concentration of agarose added to the final volume²

For these reasons we attempted to grow the L. lactis cells in a 2.5% polyacrylamide gel, a 5% acrylamide gel, a 1.5% LMP agarose gel and on the surface of a 5% polyacrylamide gel.

Half an hour in advance of making the gel, the cells were induced with nisin to start the GFP production, this way we could find out whether the cells were still viable after polymerization of the gel they were put in

The gels were observed and followed using a widefield (confocal) microscope, so far we can prove the cells are able to grow on top of a 1.5% LMP agarose gel as well as inside a 1.5% LMP agarose gel, the results are shown in figure 7

Figure 7: Cel growth inside the gel. A. After 30 minutes incubation B. After 60 minutes incubation and C. After 90 minutes incubation. And cel growth on top of the gel. D. After 30 minutes incubation E. After 60 minutes incubation and F. After 90 minutes incubation

The gel slides were then incubated overnigtht at 30 °C, to check for significant differences in growth see figure 8

Figure 8: Growth after overnight incubation at 30 C. A,B. Cells poured inside the gel. C,D. Cells on top of the gel.

Ultimately it would be the best to prove the ability of L. lactis to grow inside a gel, by making a time lapse of the growth inside a polyacrylamide gel, but for now this will remain a future prospect

References

1. Stellwagen NC., Department of Biochemistry, University of Iowa, Iowa City, USA. Apparent pore size of polyacrylamide gels: comparison of gels cast and run in Tris-acetate-EDTA and Tris-borate-EDTA buffers. Electrophoresis. (1998) volume 19 issue 10 p1542-1547.

2. Janaky Narayanan, Jun-Ying Xiong and Xiang-Yang Liu, Department of Physics, National University of Singapore, Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore. Determination of agarose gel pore size: Absorbance measurements vis a vis other techniques. Journal of Physics: Conference Series 28, (2006), p83–86.

3. Leonor Valdez-Sanchez, Lactococcus lactis, http://web.mst.edu/~microbio/bio221_2005/L_lactis.htm. Last visited: 17-10-2014.

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