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From 2010.igem.org

Alkane Sensing, Solvent Tolerance and Salt Tolerance

by Pieter

The plates containing yesterday's ligations contained colonies, to check whether they really contain the desired BioBrick a colony PCR was done, and the used colonies were grown in liquid LB medium over night. The results from the PCR were analysed on a 1% agarose gel.

• BBa_K398305 = Alks -> E0240
• BBa_K398101 = bbc1 -> J61101
• BBa_K398402 = PhPFDalpha -> J61101
• BBa_K398403 = PhPFDbeta -> J61101

Lane Description:

#	Description	Expected Length (bp)	Primers	Status	Remarks
M	SmartLadder	n/a	n/a	n/a
1	BBa_K398305	3772	G00101 + G00100	X
2	BBa_K398305	3772	G00101 + G00100	X
3	BBa_K398305	3772	G00101 + G00100	X
4	BBa_K398305	3772	G00101 + G00100	X
5	BBa_K398305	3772	G00101 + G00100	X
6	BBa_K398101	1007	G00101 + G00100	X
7	BBa_K398101	1007	G00101 + G00100	X
8	BBa_K398101	1007	G00101 + G00100	X
m	EZ Ladder	n/a	n/a	n/a
M	SmartLadder	n/a	n/a	n/a
1	BBa_K398101	1007	G00101 + G00100	X
2	BBa_K398101	1007	G00101 + G00100	X
3	BBa_K398402	836	G00101 + G00100	X
4	BBa_K398402	836	G00101 + G00100	X
5	BBa_K398402	836	G00101 + G00100	X
6	BBa_K398402	836	G00101 + G00100	X
7	BBa_K398402	836	G00101 + G00100	X
8	BBa_K398403	734	G00101 + G00100	X
m	EZ Ladder	n/a	n/a	n/a

Lane Description:

#	Description	Expected Length (bp)	Primers	Status	Remarks
M	SmartLadder	n/a	n/a	n/a
1 - 7	Eva's samples	n/a	n/a	n/a
m	EZ tLadder	n/a	n/a	n/a
1	BBa_K398403	734	G00101 + G00100	X
2	BBa_K398403	734	G00101 + G00100	X
3	BBa_K398403	734	G00101 + G00100	X
4	BBa_K398403	734	G00101 + G00100	X
M	SmartLadder	n/a	n/a	n/a

Emulsifier

When going over the plates in the fridge we noticed a few white colonies on plates from 13-07-'10. Previously we thought there were only red colonies. So we decided to do a colony PCR on all 12 white colonies.

Lane Description:

#	Description	Expected Length (bp)	Primers	Status	Remarks
M	SmartLadder	n/a	n/a	n/a
1	BBa_K398202	392	G00101 + G00100
2	BBa_K398202	392	G00101 + G00100
3	BBa_K398202	392	G00101 + G00100
4	BBa_K398202	392	G00101 + G00100
5	BBa_K398202	392	G00101 + G00100
6	BBa_K398202	392	G00101 + G00100
7	BBa_K398202	392	G00101 + G00100
8	BBa_K398202	392	G00101 + G00100
9	BBa_K398202	392	G00101 + G00100
10	BBa_K398202	392	G00101 + G00100
11	BBa_K398202	392	G00101 + G00100
12	BBa_K398202	392	G00101 + G00100

Alkane degradation

For the next step in BioBrick formation a digestion was done:

#	Sample	Enzyme 1	Enzyme 2	Enzyme 3	Buffer	BSA	Needed fragment
1	1 μg 007T	EcoRI	SpeI	BamH1	2 (Biolabs)	✓	‘E – J61100-AlkB2 – S’
2	1 μg 008A	EcoRI	XbaI		2 (Biolabs)	✓	‘E – J61100-rubA3 – X’
3	1 μg 010A	EcoRI	SpeI	AseI	2 (Biolabs)	✓	‘E – J61100-rubR – S’
4	2 μg B0015	EcoRI	XbaI		2 (Biolabs)	✓	‘E – B0015 – pSB1AK3 – X’
5	1 μg 017A	EcoRI	SpeI	AseI	2 (Biolabs)	✓	‘E – J61100-ladA – S’
6	1 μg 018A	XbaI	PstI	AseI	2 (Biolabs)	✓	‘X – J61101-ADH – P’
7	1 μg 019A	EcoRI	SpeI	AseI	2 (Biolabs)	✓	‘E – J61107-ALDH – S’
8	1 μg pSB1K3	EcoRI	PstI		2 (Biolabs)	✓	‘E – pSB1K3 – P’

Results of the digestion on 1% agarose gel

The gel was runned at 100V for 1 hour, loaded 5 μL of marker and 5 μL sample + 1 μL loadingbuffer

Lane description:

#	Description	Expected Length (bp)	Status
1	Smartladder
2	‘E – J61100-AlkB2 – S’ (007T)		✗
3	Undigested 007T		✗
4	‘E – J61100-rubA3 – X’ (008A)		✓
5	Undigested 008A		✓
6	‘E – J61100-rubR – S’ (010A)		✓
7	Undigested 010A		✓
8	‘E – B0015 – pSB1AK3 – X’		✓
9	pSB1AK3-B0015		✓
10	‘E – J61100-ladA – S’ (017A)		✓
11	Undigested 017A		✓
12	‘X – J61101-ADH – P’ (018A)		✓
13	Undigested 018A		✓
14	‘E – J61107-ALDH – S’ (019A)		✓
15	Undigested (019A)		✓
16	‘E – pSB1K3 – P’		✓
17	Smartladder