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From 2010.igem.org

Contents 1 Alkane Sensing, Solvent Tolerance and Salt Tolerance 1.1 PCR 1.2 Digestion 1.3 Ligation

Alkane Sensing, Solvent Tolerance and Salt Tolerance

by Pieter

I tried to assemble the remaining biobricks from various sub projects. The goal is to assemble the following:

• AlkS + E0240
• PalkB + J06702
• J61101 + bbc1
• J61101 + PhPFDalpha
• J61101 + PhPFDbeta

To achieve this J06702, bbc1, PhPFDalpha and PhPFDbeta were amplified by PCR. The J61101 was cut open with S and P after which the biobricks were inserted. AlkS was inserted in the backbone with E0240.

PCR

The PCR protocol was followed for the amplification of the BioBricks.

PCR reactions

#	Description	Expected lenght (bp)	Primer	Status	Remarks
1	Marker	n/a	n/a	n/a
2	J06702	869	G00100 + G00101	✗
3	bbc1	703	MF + MR2	✓
4	PhPFDalpha	528	MF + MR2	✓
5	PhPFDbeta	430	MF + MR2	✓

Digestion

The PCR products were subsequently digested:

#	Sample	Enzyme 1	Enzyme 2	Buffer	BSA	Needed fragment
1	2 μL AlkS	EcoRI	SpeI	2 (BioLabs)	✓	‘E–AlkS–S’
2	3 μL E0240	EcoRI	XbaI	2 (BioLabs)	✓	‘X–E0240–E’
3	16 μL PalkB	SpeI	PstI	2 (BioLabs)	✓	‘P–PalkB–S’
4	40 μL J06702 PCR product	XbaI	PstI	2 (BioLabs)	✓	‘X–J06702–P’
5	6 μL J61101	SpeI	PstI	2 (BioLabs)	✓	‘P–J61101–S’
6	40 μL bbc1 PCR product	XbaI	PstI	2 (BioLabs)	✓	‘X–J61101–P’
7	40 μL PhPFDalpha PCR product	XbaI	PstI	2 (BioLabs)	✓	‘X–PhPFDalpha–P’
8	40 μL PhPFDbeta PCR product	XbaI	PstI	2 (BioLabs)	✓	‘X–PhPFDalpha–P’

Ligation

The digestion products were ligated over two nights:

#	BioBrick	Fragment 1	Fragment 2	Final volume
1	AlkS + E0240	10 μL ‘E–AlkS–S’	10 μL ‘X–E0240–E’	25 μL
2	J61101 + bbc1	7 μL ‘P–J61101–S’	1 μL ‘X–J61101–P’	10 μL
3	J61101 + PhPFDalpha	7 μL ‘P–J61101–S’	1 μL ‘X–PhPFDalpha–P’	10 μL
4	J61101 + PhPFDbeta	7 μL ‘P–J61101–S’	1 μL ‘X–PhPFDbeta–P’	10 μL