Light transmission of solid agar, different agar concentrations and growth media

Purpose:

In order to create the 3D-Printer based on a double-light-sensitive-promotor, it is not only necessary to immobilize the cells within a solid medium but also to be able to induce the protein expression at a definied spot. Thus, we want to examine the light transmission properties of different solid media.

Procedure:

To gain data on the light transmission of different solid media, different compositions of media were produced and poured into 1 ml cuvettes while they were still liquid. After gelation, a transmission spectrum (230 nm - 750 nm) was recorded for each sample

Agar with LB-Medium:

Agar with M9-Medium:

Agarose with M9-Medium:

Is a glas or plastic container neccessary for solid media?

Purpose:

We figured that if we could do without a glas or plastic container surrounding the solid medium, this would increase the precision of our light-beam aiming at the cells that are to be induced (no light refraction at the container walls, etc.).

Procedure:

To examine if it is possible to make a detachted, self-supporting block of agar, we made a 40 ml block of 0.5 % (w/v) Agar with M9-Medium in a small beaker glass. After the gellation process we turned it out and left it to stand over night.

Alternative growth media and gelling agents

Purpose:

The purpose of this part of the experiment is to test different gelling agents known from everyday life. As it had turned out in experiment 1.1, a very clear gel is required. From cooking, we know that gelatine and jelly glaze provide quite clear gels.

Procedure:

Three different gelling agents were tested:

1) "Küchle Blitzguss", a jelly glaze that doesn't require heating

2) "RUF Tortenguss klar", a normal jelly glaze that has to be boiled

3) Gelatine

For each substance, three different concentrations were prepared M9:

1) according to the recipe

2) with half the amount of liquid added

3) with twice the amount of liquid added

For each of these, three cuvettes were prepared and a transmission spectrum was recorded after gelation.

Mixing gelling agents with bacterial cultures

Testing of 5 different gelling agents for O2-permeability and stability mixed with B. subtilis and E. Coli. Agents were mixed with 9 ml of LB and 1 ml of liquid culture and incubated at 37° C o/n.

1) Agar-Agar 0.5 %

2) Gelatine 1.8 % (w/v)

3) "Blitz-Guss" 5.9 % (w/v) cold

4) "Tortenguss" 0.9 % (w/v) warm

5) Agarose 0,5 %

testing of alternative gelling agent: GELRITE

0,5% GELRITE in M9-medium gelled at 47° C. Clear and quite stable gel. 0,25% GELRITE in M9-medium gelled at 40° C. Clear and stable gel. not stable enough without plastic borders.

light scattering in gels with E. Coli

0.5 % Agarose

0.5 % Agar-Agar

0.5 % GELRITE

18 ml of gelling agent was heated, cooled down to 40° C, poured into a petri dish with 1 ml AHL (final concentration in petri dish: 100 nM) and 1 ml E. Coli (express GFP with inducer AHL) culture in LB.

GELRITE gelled immediately upon contact with cold petri dish. --> all components have to be warmed to aprox. 50° C

light scattering in gels

0.25 % GELRITE was produced: 25 ml M9 and 0,15 g GELRITE was heated and then mixed with 5 ml of a dense E. Coli suspension (OD = ca. 10) in M9 and poured into a cuvette. Transmission was measured with a absorption photometer.

Quick gelling of GELRITE was again a problem. Spectra showed higher transmissions in blue and UV ranges than Agarose and Agar-Agar spectra before. A very high density of cell culture lead to a decrease in transmission of about 95%. Media:0,25%_Gelrite_with-without_cells.pdf‎

---> Cells lead to a great decrease in light trasmission ---> we have to quantify this

Findings from literature: M9 shouldn't be used in a lower concentration than 1 x to ensure growth of E. coli. However, 1x M9 includes a lot of salt, which leads to early gelling of GELRITE (0,5 % GELRITE (w/v) at 47° C in 1 x M9)

-> testing of different GELRITE concentrations with 1x M9

0,1 % GELRITE with stirring: gelling at 43 - 44° C, however rather sirupy due to stirring 0,1 % GELRITE without stirring: gelling at 44° C, however not a very stable gel

-> for a somewhat stable gel, 44° C is the lowest temperature in 1x M9 and GELRITE

repetition with 0,5 % Agarose: gelling at 30° C and transmission spectrum taken -> low gelling temperature but murky gel

permeability of gels

0,5 % Agarose (about 10 ml) in a plastic syringe with blue water (with ink) on the top, without and with pressure -> no water drops out of the syringe, even when pressure is applied (rather the gel is pressed out) -> no permeability for water

0,5 % GELRITE in the same setup over night: no permeability for water, ink diffused into the gel

inoculation of 30 ml LB medium with E. coli (BW27785) and used for experiments on 18-04-2011

Testing of Silica Sol-Gels

according to "Immobilization of bacteria in silica matrices using citric acid in the sol–gel process" Alvarez et al. (DOI 10.1007/s006-00253-0580-6)

6 ml of ddH2O were added to 1g silica and heated to 80° C untill full colloidal suspension was achieved. The suspension was cooled to room temperature und either pH 6.5 was set with 0.75 M citric acid or the suspension was mixed 1:1 with 1.8 x 10^8 cfu/ml E.Coli. After 2 minutes the gelling should occur.

Testing of Silica Sol-Gels II

The experiment from 18-04-2011 was repeated using Natronwasserglas (silicic acid) and silicon dioxide (Kieselgel 60) according to Alvarez et al with the same results as above.

Adjustment of E. Coli cell count in GELRITE-Gels

Aim: maximum cell count determination as regards to transmission of visible light

Preparation of 0.5 % GELRITE-gels

0.15 g GELRITE were added to 25 ml M9-medium. The solution was heated to aproximately 90° C (or 100% of GELRITE was dissolved) and cooled down to 50° C in a water bath. 5 ml bacterial suspension with apropriate cell count was heated to 50° C and poured imediately into the GELRITE solution. Mixture was stirred thoroughly and poured into petri dishes or cuvettes. Gelling should occur immediately after contact with cold plastic surface. (at 47° C)

Following in-gel cell counts were produced:

1x10⁷ cells/ml: pic spectrum
1x10⁸ cells/ml: pic spectrum
1x10⁹ cells/ml: pic spectrum

Media:Variation_of_cell_density 04-11-19.jpg

results:

Cell count has to be somewhere between 1x10⁸ and 1x10⁹ cells/ml in regards to efficient transmission. Space between single E. Coli cells is to high.

Adjustment of E. Coli cell count in GELRITE-Gels II

A new culture of E. Coli BW27783 with Nalidixin acid resistance (Nx) was thawed (Stock 5 OD 3,66) and transferred to 45 ml LB with 50 µg/ml Nx (1:1000 dilution of stock 50 mg/ml).

starting OD: 0,03 (10:30)

0.5 % GELRITE-gels were produced as described above (19-04-2011, Preparation of 0.5 % GELRITE-gels).

Following in-gel cell counts were produced:

2x10⁸ cells/ml: pic spectrum
4x10⁸ cells/ml: pic spectrum
6x10⁸ cells/ml: pic spectrum
8x10⁸ cells/ml: pic spectrum

Media:Variation_of_cell_densities.jpg

Viability Determination of E. Coli in GELRITE-Gels

aim: Determination of viable cell count(vcc) after 50° C shock in gel production process

vcc of culture: dilution series and plating on LB plates.

vcc of gels: 0.1g of GELRITE slices were homogenized in 0.9 ml LB and plated on LB plates.

gel with more components

Testing bacteria in the Gelrite matrix with more medium components:

- heat resistant E. coli BH28 under Kan resisitance

- positive control with lacZ, induceable with Arabinose, under Amp resistance

0,35 % Gelrite (for delayed gelling -> did't change so much, gelling at 47 °C)

M9 medium

1 mg/ml ammonium iron sulfate

2 mg/ml arabinose

-> cooked to dissolve components

bacteria (final concentration in the gel 1*10^7 bact./ml)

added with the bacteria / not cooked / heated to ca. 50 ° C in water bath: 0,3 mg/ml S-Gal (from 50 mg/ml stock in DMF -> stock can be found in our freezer, brownish liquid in eppi)

antibiotic 100 µg/ml Medium

problems:

- premature gelling

- blurry gel

- light brownish color (like S-Gal in DMF)