From 2010.igem.org

Lab work

Alkane Sensing, Solvent Tolerance, Salt Tolerance, Emulsifier

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

The digestion products were ligated in the afternoon:

#	BioBrick	Fragment 1	Fragment 2	Final volume
1	J61101 + bbc1	6 μL ‘X – bbc1 - P’	6 μL ‘P – J61101 - S’	20 μL
2	J61101 + PhPFDalpha	6 μL ‘P – J61101 – S’	6 μL ‘X – PhPFDalpha – P’	20 μL
3	J61101 + PhPFDbeta	6 μL ‘P – J61101 – S’	10 μL ‘X – PhPFDbeta – P’	20 μL
4	R0011-B0032 (9) + OprG-B0015	10 μL ‘P – R0011 - B0032 – S’	4 μL ‘X – OprG - B0015 – P’	20 μL
5	R0011-B0032 (12) + OprG-B0015	10 μL ‘P – R0011 - B0032 – S’	4.3 μL ‘X – OprG - B0015 – P’	20 μL
6	R0011-B0032 (9) + AlnA-B0015	10 μL ‘P – R0011 - B0032 – S’	4.3 μL ‘X – AlnA - B0015 – P’	20 μL
7	R0011-B0032 (9) + AlnA-B0015	10 μL ‘P – R0011 - B0032 – S’	4.3 μL ‘X – AlnA - B0015 – P’	20 μL

Transformation

10 μL of ligation mixes 1 - 3 were transformed Top10 competent cells, and these were grown on LB-plates with antibiotic Amp over night.

10 μL of ligation mixes 4 - 7 were transformed DH5alpha competent cells, and these were grown on LB-plates with antibiotic Tet over night.

Alkane degradation

Today we started with a PCR of a number colonies from yesterday's transformants. These had all given colonies on the plates, so hopefully they also contain the BioBricks! Five colonies from each plate were taken for the colony PCR, and after the PCR the products were analyzed on two gels.

Of BioBricks 007A, 012K, 021A and 021K there were a number of successful PCRs. Overnight an extra PCR will be done on some extra colonies from 020A. This was the only 3-way ligation done, which is probably why it's the only one that was unsucessful. The reason 011A failed was because the BrioBrick used to create this (007T) was wrong after all (seen during the digestion check of yesterday

PCR 1

Lane description

#	Description	Primers	Expected length (bp)	✓	✗
L	SmartLadder	n/a	n/a	n/a	n/a
1-5	007A	G00100 + G00101	1538	1,2,4	3,5
6-10	011A	G00100 + G00101	1816	none	6,7,8,9,10
11-15	012K	G00100 + G00101	1720	3,5	1,2,4
L	SmartLadder	n/a	n/a	n/a	n/a

PCR 2

Lane description

#	Description	Primers	Expected length (bp)	✓	✗
L	SmartLadder	n/a	n/a	n/a	n/a
1-5	020K	G00100 + G00101	2428	none	1,2,3,4,5
6-10	021A	G00100 + G00101	2011	8,10	6,7,9
11-15	021K	G00100 + G00101	2011	11,12,13,15	14
16	021KA	G00100 + G00101	2011	✓
L	SmartLadder	n/a	n/a	n/a	n/a

Hydrocarbon sensing

PCR Amplification

To assembly the small parts like the promoters to the RBS were performed an overlapping PCR. For the parts that have to be connected to each other, primers were designed with a overlapping overhang. Then the parts were amplified using primers with the overhang. The product was put on 1% agarose gel:

Lane description:

#	Description	Primers	Expected length (bp)	Staus
1	1 μL PalkS12	MF + 304R
2	1 μL B0032	304F + G00101
3	1 μL P(Caif)	MF + 327R
4	1 μL B0032	327F + G00101

Team:TU Delft/4 August 2010 content