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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.

• BBa_K398202 = R0011 + B0032

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		Isolated plasmid
10	BBa_K398202	392	G00101 + G00100
11	BBa_K398202	392	G00101 + G00100
12	BBa_K398202	392	G00101 + G00100		Isolated plasmid

None of the bands is truly positive. Still number 4,5 and 9 could be positive. Or due to the GC content the bands could be shifted a little bit upwards, so that maybe even sample 12 could be positive.

Just to be sure, we isolated the plasmids and send them to BaseClear for sequencing. The results will be in by Thursday.

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