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Contents 1 Lab 208 1.1 Main purpose 1.2 Who was in the lab 1.3 Procedure 1.4 Results 1.5 Conclusion 2 lab 115 2.1 Main purpose 2.2 Who was in the lab 2.3 Procedure 2.4 Results 2.4.1 Colorimetric results 2.4.2 OD at the end of the experiment 2.5 Conclusion

Lab 208

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Main purpose

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Who was in the lab

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Henrike, Kristian, Julia

Procedure

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A gradient PCR was made for pSB1C3 and pZA21::ara for Nir.

The master mix contains:

• CG buffer,
• X7 polymerase,
• 2% of DMSO (1uL of 100%DMSO) per reaction
• 1mM MgCl2 (1uL of 50mM MgCl2) per reaction

	1	2
Template	pSB1C3	pZA21::ara
Primers	57a + 57b	58a + 58b
Temperature range	58C-78C	50C-65C
Elongation time per cycle	1:30 min	2 min

Each reaction was run in one of the 12 different annealing temperatures within the range indicated in the table. We followed the standard PCR program.

Results

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Conclusion

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lab 115

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Main purpose

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Run Experiment 4 in two different samples aerobically in order to characterize the behavior of HAO transformants and the Nitrosomonas europaea strain.

Who was in the lab

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Ariadni, Helen, Kashia

Procedure

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Adjusting the temperature at 36 degrees and calibrating the probes as described in Calibration protocol.

Following the protocol Experiment 4

Growing:

• two replicates of 4 ml from HAO overnight culture in 50 ml of DM minimal medium with NH₄Cl and 25 ul of hydroxylamine solution
• 4 ml from Nitrosomonas Europaea in 42 ml of ATCC media with 20 ul of hydroxylamine solution
• 4 ml from Nitrosomonas Europaea in 44 ml of ATCC media with 0.7 ml of ammonium solution

OD was measured OD=0.4017 and 0.3864 for the HAO mutant. 0.0822 and 0.0947. For Nitrosomonas Europaea was 0.0095 and 0.0084.

Results

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Colorimetric results

Ranges

• Measuring range 5-150 mg/L NH₄-N
• Measuring range 0.02-1 mg/L NO₂-N

Standard solutions

• Ammonium - 66.6 mg/L (expected 39 mg/L)
• Nitrite - 0.72 mg/L (expected 0.5 mg/L)

time (min)	ammonium AMO1(mg/L)	ammonium AMO2(mg/L)	Ammonium E.coli(mg/L)	nitrite E.coli	ammonium abiotic (mg/L)	nitrite abiotic(mg/L)
0	<5	<5	<5	<0.02	5	<0.02
13	<5	<5	5	<0.02	-	-
19	<5	<5	5	<0.02	-	-
spike	146	140	<5	<0.02	-	-
35	158	244	258	<0.02	-	-
41	169.5	171	178	<0.02	-	-
52	226	280	250	<0.02	-	-
64	232	254	124	<0.02	-	-
90	238	272	246	<0.02	-	-
10 hours	172	218	16	<0.02	154	<0.02

OD at the end of the experiment

OD=0.2083 and 0.2232 for the AMO mutants. For the E.coli culture the OD was 2.39 and 0.1173.The abiotic controls have OD=0.00144 and 0.0004.

Conclusion

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