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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

Lab 208

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Main purpose

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PCR to amplify pSB1C3 and pZA21::ara for Nir

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, Kasia

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)	nitrite HAO1(mg/L)	nitrite HAO2(mg/L)	Ammonium N.Europaea(mg/L)	nitrite N.Europaea
0	<0.02	<0.02	0	<0.02
6	<0.02	<0.02	0	<0.02
13	<0.02	<0.02	0	<0.02
spike	<0.02	<0.02	246	<0.02
25	<0.02	<0.02	250	<0.02
39	<0.02	<0.02	278	<0.02
105	<0.02	<0.02	266	<0.02
19 hours	<0.02	<0.02	220	<0.02

Note: there was some more nitrite for N.Europaea but it was still less than <0.02 mg/L at the end of the experiment

OD at the end of the experiment

OD( HAO1)=0.28 and OD(HAO2)=0.278. For the N.Europaea culture the OD was 0.1348 and 0.0114 for the solution with ammonium and for the one with hydroxylamine, respectively.

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