Team:LMU-Munich/Data

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The LMU-Munich team is exuberantly happy about the great success at the World Championship Jamboree in Boston. Our project Beadzillus finished 4th and won the prize for the "Best Wiki" (with Slovenia) and "Best New Application Project".

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Data

Here you will find all of our project data. For our big breakthroughs, or to follow specific projects, see the individual project pages:

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Bacillus Intro	Bacillus BioBrickBOX	SporeCoat FusionProteins	Germination STOP

Bacillus BioBrick Box - Promoters

Fig. 1: Luminescence measurement of Anderson promoters in the reporter vector pSB_Bs3C-luxABCDE. OD₆₀₀ (right), LUMI (center) and OD₆₀₀ per LUMI (left) depending on the time (h) are shown for two different clones (green/blue). Data derive from three independent experiments. Curves were fitted over each other (t=0, OD₆₀₀=0,3) and smoothed by taking average of three neighboring values.

Eleven of the nineteen promoters of the [http://partsregistry.org/Part:BBa_J23100 Anderson collection] (J23100,J23101, J23102, J23103, J23106, J23107, J23113, J23114, J23115, J23117, J23118) were evaluated in the reporter vector pSB_Bs3C-luxABCDE from the BioBrickBox containing the lux operon as a reporter for promoter activity. The gene expression which correlates to the promoter activity leads to the expression of the lux operon with the luciferase. The luminescence which is produced by the luciferase can be measured with the plate reader (BioTek). Data derive from three undependant measurements (Fig. 1). Curves were fitted over each other (t=0, OD₆₀₀=0,3) and smoothed by taking average of three neighboring values. OD₆₀₀ values shown are plate reader units and about one third of the usual OD₆₀₀ values. All clones show a usual growth curves. The activity of the promoters raises during the pass from the transition to the stationary phase. This maximum (t=1h) reaches from 200Lumi/OD₆₀₀ (promoter J23115) to a maximum of 1500 Lumi/OD₆₀₀ for the strongest promoter (J23101). Afterwards the activity goes down to the beginning level (t=2h). The oscillation of luminescence (Lumi/ OD₆₀₀ in the beginning of the curves are due to the small OD₆₀₀ and do not mean a high promoter activity. The luminescence of one clone of the promoters J23107 and J23114 do not show activity where in future a second clone with promoter activity should be measured. In comparison to all the other evaluated Bacillus promoters these Anderson promoters showed a very low acitivity in B. subtilis.

To measure the activity not only with the lux reporter operon, four promoters of the Anderson collection were cloned into the reporter vector pSB_Bs1C-lacZ to do β galactosidase assays and then to compare the results of the strength of these promoters in B. subtilis. (Fig. 2)

Fig. 3: Luminescence measurement of the constitutive Bacillus promoters P_liaG and P_lepA in the reporter vector pSB_Bs3C-luxABCDE. OD₆₀₀ (up), LUMI (center) and LUMI per OD600 (down) depending on the time (h) are shown for two different clones (green/blue). Data come from three independent experiments. Curves were fitted over each other (t=0, OD600=0,3) and smoothed by taking average of three neighboring values.

The constitutive promoters P_liaG and P_lepA were evaluated in the reporter vector pSB_Bs3C-luxABCDE which contains the lux operon. Data derive from three undependant measurements (Fig. 3). Curves were fitted over each other (t=0, OD₆₀₀=0,3) and smoothed by taking average of three neighboring values. OD₆₀₀ values shown are plate reader units and about one third of the usual OD values. All clones show a usual growth curves. The activity of the promoters raises during the pass from the transition to the stationary phase. The second clone of the promoters P_lepA and P_liaG did not show any luminescence activity. In the beginning of the growth curve the activity of both promoters raise to their maximum. They show a similar behaviour in pertaining to the groth curve. P_liaG has an activity maximum of about 100.000 Lumi/OD₆₀₀ during the pass from logarithmic to the stationary phase. P_lepA shows an maximum of about 400.000 Lumi/OD₆₀₀. Comparing these two constitutive promoters the activity of P_lepA is about four times higher than the activity of P_liaG. In the late stationary phase the activity completely disappears.

Fig. 4: β galactosidase assay and growth curve P_liaG (black) and P_veg (grey) in the reporter vector pSB_Bs1C-lacZ. β galactosidase activity (Miller Units)and growth curve are the average of two independant clones. Experiment shows representative data which was obtained in the same way from three independent experiments.

The β galactosidase assay of the constitutive Bacillus promoters Pveg and P_liaG was repeated three times. Data show one representative result. Two undependant clones of B. subtilis with the same construct were measured and their mean with standard deviation is show in the graph. In the beginning of the growth curve both promoters show a small activity. But then it raises to a maximum before it decreases to the begininng level after about seven hours (Data not shown). Summing up the course of activity of both promoters P_veg and P_liaG is very similar based on the growth curve. The highest beta galactosidase activity and therefore the highest activity of the promoter Pveg can with an maximum of 65 Miller units be found during the transfer from the logarithmic to the stationary phase. This is about five times higher than the acitivity of the promoter P_liaG with an maximum activity of about 12 Miller Units.

Fig. 5:

Fig. 6:

Sporobeads

To purify our spores from the vegetative cells we treated the samples, that were grown for 24 hours in DS-Medium, with three different methods. The data is summarized in the following table:

	all cells	mature spores	immature spores

untreated wildtype	7.29 x 10⁸ /ml	1 x 10⁸ /ml 13.71%	0.04 x 10⁸ /ml 0.55%
untreated P_cotYZ-cotZre-gfp-terminator	6.79 x 10⁸ /ml	1 x 10⁸ /ml 14.72%	0.13 x 10⁸ /ml 1.9%
French Press wildtype	4.87 x 10⁸ /ml	2.1 x 10⁸ /ml 43%	0.05 x 10⁸ /ml 1%
French Press P_cotYZ-cotZre-gfp-terminator	4.75 x 10⁸ /ml	1.88 x 10⁸ /ml 39.58%	0.05 x 10⁸ /ml 1%
Sonification wildtype	4.6 x 10⁸ /ml	1.22 x 10⁸ /ml 26.52%	0.1 x 10⁸ /ml 2%
Sonification P_cotYZ-cotZre-gfp-terminator	6.72 x 10⁸ /ml	1.53 x 10⁸ /ml 22.77%	0.23 x 10⁸ /ml 3%
Lysozyme wildtype	2.48 x 10⁸ /ml	1.58 x 10⁸ /ml 63.7%	0 /ml
Lysozyme P_cotYZ-cotZre-gfp-terminator	1.05 x 10⁸ /ml	1.05 x 10⁸/ml 100%	0 /ml

Tab. 1 shows that after treatment with French Press and ultrasound the number of spores compared to the untreated samples was increased. We assume the reason for this was the impurity of these samples that derived from damaged vegetative cells. Thus, during counting it was not always possible to distinguish between mature spores and cell waste. However, a huge difference in number of vegetative cells and spores between untreated and lysozyme treated samples was noticeable under microscopy as it is visualized in the table above and the pictures below. Because the vegetative cells were lyzed and not damaged it was easy to recognize the mature spores.

Germination Stop

We induced our germination-mutant strains to sporulate in Difco sporulation media. Then we measured the germination rate of mutant spores in a germination assay.

Fig. 5: Comparison of colony growth on LB-agar (plus antibiotics) plates. Upper plates are plated with DSM cell/spore cultures that have been heated at 80°C for 1 hour to kill living cells (but does not affect spores). This prevents cells from forming colonies. Therefore, all colonies observed are from germinated spores. Lower plates contain cultures which have not been heated. Growth on these plates is from either cells or spores, and demonstrates that strains are able to grow. WT168 has no germination knockouts; strains B40-B43 have triple knockouts; strains B46 and B47 have quadruple knockouts; Spo0A has a knockout of the sporulation gene Spo0A (replaced with a tet cassette). WT168 is a positive control; Spo0A is a negative control.
Strains:
B40 -- cwlD::kan, sleB::mls, cwlJ::spec
B41 -- cwlD::kan, sleB::mls, gerD::cat
B42 -- cwlD::kan, cwlJ::spec, gerD::cat
B43 -- gerD::cat, sleB::mls, cwlJ::spec
B46 -- cwlD::kan, cwlJ::spec, gerD::cat, sleB::mls
B47 -- gerD::cat, sleb::mls, cwlJ::spec, cwlD::kan

The plate growth demonstrates the inability of our mutant spores to germinate. We can say that fewer than 1 out of 3x10^7 spores of strains B40, B41, B43, B46, and B47 germinated.

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-	<p align="justify">Tab. 1 shows that after treatment with French Press and ultrasound the number of spores compared to the untreated samples was increased. We assume the reason for this was the impurity of these samples that derived from damaged vegetative cells. Thus, during counting ~~we were~~ not ~~able~~ to distinguish between mature spores and cell waste. However, a huge difference in number of vegetative cells and spores between untreated and lysozyme treated samples was noticeable under microscopy as it is visualized in the table above.	+	<p align="justify">Tab. 1 shows that after treatment with French Press and ultrasound the number of spores compared to the untreated samples was increased. We assume the reason for this was the impurity of these samples that derived from damaged vegetative cells. Thus, during counting it was not always possible to distinguish between mature spores and cell waste. However, a huge difference in number of vegetative cells and spores between untreated and lysozyme treated samples was noticeable under microscopy as it is visualized in the table above and the pictures below. Because the vegetative cells were lyzed and not damaged it was easy to recognize the mature spores.
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