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From 2010.igem.org

Contents 1 Groningen Team 2 Lab Work 2.1 Alkane Sensing, Solvent Tolerance and Salt Tolerance 2.2 Alkane degradation 2.2.1 Colony PCR 2.2.2 Plasmid Isolation 2.2.3 Digestion 2.2.4 Ligation 2.2.5 Transformations

Groningen Team

Today three members had an appointment with one of the professors at our department. We heard of this and invited them to come over to meet each other.

Lab Work

Alkane Sensing, Solvent Tolerance and Salt Tolerance

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.

Lane Description:

#	Description	Expected Length (bp)	Primers	Status	Remarks
1	SmartLadder	n/a	n/a	n/a
2	BBa_K398101	929	G00101 + G00100	✓
3	BBa_K398101	929	G00101 + G00100	✗
4	BBa_K398101	929	G00101 + G00100	✗
5	BBa_K398101	929	G00101 + G00100	✗
6	BBa_K398101	929	G00101 + G00100	✗
7	BBa_K398402	758	G00101 + G00100	✗
8	BBa_K398402	758	G00101 + G00100	✗
9	BBa_K398402	758	G00101 + G00100	✓
10	SmartLadder	n/a	n/a	n/a
11	BBa_K398402	758	G00101 + G00100	✗
12	BBa_K398402	758	G00101 + G00100	✗
13	BBa_K398403	656	G00101 + G00100	✗
14	BBa_K398403	656	G00101 + G00100	✗
15	BBa_K398403	656	G00101 + G00100	✗
16	BBa_K398403	656	G00101 + G00100	✓
17	BBa_K398403	656	G00101 + G00100	✗

Alkane degradation

Colony PCR

The morning started with a gel checking the PCR products of the one BioBrick that didn't give positives yesterday. This gel showed one positive, but when I centrifuged the cells, they gave a red pellet. This indicated that the plasmid unfortunately was not the desired BioBrick, but the original destination plasmid, so we'll have to try again.

Lane description:

#	Description	Primers	Expected length (bp)	✓	✗
L	SmartLadder	n/a	n/a	n/a	n/a
1-9	020K	G00100 + G00101	2428	6	rest

Plasmid Isolation

Of the colonies which gave a positive PCR yesterday, the plasmids were isolated using a QIA-gen miniprep kit. The following concentrations were obtained:

BioBrick	Composed of	Concentration (ng/μL)
007A (1)	J61100-alkB2	33.8
007A (2)	J61100-alkB2	32.0
012K	J61100-rubR-B0015	42.7
021A	J61107-ALDH-B0015	46.3
021K (5)	J61107-ALDH-B0015	7.3
021K (1)	J61107-ALDH-B0015	11.1
021K (2)	J61107-ALDH-B0015	12.9

For some reason the 021K plasmid simply didn't want to be purified. All three of the isolates had a very low concentration. Next week we'll try another plasmid isolation method and see if that works.

Digestion

A few of the plasmids just isolated were digested:

#	Sample	Enzyme 1	Enzyme 2	Enzyme 3	Buffer	BSA	Needed fragment
1	009A	EcoRI	SpeI	PvuI	2 (Biolabs)	✓	‘E - J61100 - rubA4 - S’
2	012K	EcoRI	XbaI	✗	2 (Biolabs)	✓	‘E – J61100 - rubR - B0015 – X’
3	007A	EcoRI	SpeI	PvuI	2 (Biolabs)	✓	‘E – J61100 - alkB2 – S’
4	008A	EcoRI	XbaI	✗	2 (Biolabs)	✓	‘E – J61100 - rubA3 – X’

The digestions were checked on a gel:

Lane description

#	Description	Expected size (bp)	OK?	#	Description	Expected size (bp)	OK?
L	Smartladder (3μl)	n/a	n/a	6	009A cut	267	✓
1	007A cut	1323	✓	6	009A uncut	n/a
2	007A uncut	n/a		7	012K cut	3552	✓
3	008A cut	2640	✓	8	012K uncut	n/a
4	008A uncut	n/a

Ligation

Following the digestions, the fragments were [Team:TU_Delft/protocols/ligation|ligated]] for 4 hours.

#	BioBrick	Fragment 1	Recipient vector
1	013AK	15 μL ‘E – J61100 - rubA4 – S’	15 μL ‘X – J61100 - rubR - B0015 - pSB1AK3 – E’
2	011A	15 μL ‘E – J61100 - alkB2 – S’	15 μL ‘X – J61100 - rubR - B0015 - pSB1AK3 – E’

Transformations

With the ligation mixes we transformed Top10 competent cells, and grew them overnight on solid LB plates.