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From 2010.igem.org

Contents 1 Alkane Sensing, Solvent Tolerance and Salt Tolerance 1.1 Digestion 1.2 Ligation 1.3 Transformation 2 Emulsifier

Alkane Sensing, Solvent Tolerance and Salt Tolerance

by Pieter

We found it remarkable that the only positive samples we found so far were from the series without preamplifying the insert by PCR (e.g. the AlkS). This is why we decided to retry the insertion of bbc1, PhPFDalpha and PhPFDbeta in J61101, but this time without PCRing the insert.

Digestion

For the assembly of BBa_K398101, BBa_K398205, BBa_K398206, BBa_K398402 and BBa_K398403 a digestion was done:

#	Sample	Enzyme 1	Enzyme 2	Enzyme 3	Buffer	BSA	Needed fragment
1	1 μg bbc1	XbaI	PstI	ScaI	2 (Biolabs)	✓	‘X – bbc1 - P’
2	1 μg PhPFDalpha	XbaI	PstI	ScaI	2 (Biolabs)	✓	‘X – PhPFDalpha - P’
3	1 μg PhPFDbeta	XbaI	PstI	ScaI	2 (Biolabs)	✓	‘X – PhPFDbeta - P’
4	1 μg J61101	SpeI	PstI		2 (Biolabs)	✓	‘S – J61101 - P’
5	1 μg OprG-B0015	XbaI	PstI		2 (Biolabs)	✓	‘X – OprG-B0015 - P’
6	1 μg AlnA-B0015	XbaI	PstI		2 (Biolabs)	✓	‘X – AlnA-B0015 - P’
7	1 μg R0011-B0032 (sample 9)	SpeI	PstI		2 (Biolabs)	✓	‘S – R0011-B0032 - P’
8	1 μg R0011-B0032 (sample 10)	SpeI	PstI		2 (Biolabs)	✓	‘S – R0011-B0032 - P’

Ligation

Transformation

Emulsifier

While waiting for the sequencing results of BBa_K398202 we already proceed with the insertion of OprG and AlnA.

Digestion

Ligation

Transformation