From 2010.igem.org

Lab work

Alkane degradation

Colony PCR

Friday, a colony PCR on colonies from a plate with transformants with BioBrick 013AK did not give positive results. But this doesn't mean that none of the colonies are correct. To check we did another colony PCR today on 8 more colonies of the AMP plate as well as the KAN plate. Hopefully a the PCR product of a colony will show the correct length.

Lane description

#	Description	Primers	Expected length (bp)	✓	✗
L	SmartLadder	n/a	n/a	n/a	n/a
1-8	013A	G00100 + G00101	1929	none	all
9-16	013K	G00100 + G00101	1929	none	all
L	SmartLadder	n/a	n/a	n/a	n/a

Unfortunately none of the PCR products seemed to have the correct length. We'll try to make the BioBrick again.

Digestion, Ligation, Transformation

A digestion was done on a number of BioBricks for the production of new BioBricks.

#	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	017A	SpeI	PstI		2 (Biolabs)	✓	‘P - pSB1A2 – J61100 - ladA – S’
4	018A	XbaI	PstI	PvuI	3 (Biolabs)	✓	‘X – J61107 - ALDH – P’

The digestion was checked on a gel, together with the uncut BioBricks:

Lane description

#	Description	Expected size (bp)	OK?	#	Description	Expected size (bp)	OK?
L	Smartladder (3μl)	n/a	n/a	5	017A cut
1	009A cut			6	017A uncut	n/a
2	009A uncut	n/a		7	018A cut
3	012K cut			8	018A uncut	n/a
4	012K uncut	n/a

For some samples a too low concentration was loaded, but furthermore the digestions looked ok.

Ligation

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

#	BioBrick	Fragment 1	Recipient vector
1	013AK	10 μL ‘E – J61100 - rubA4 – S’	10 μL ‘X – J61100 - rubR - B0015 - pSB1AK3 – E’
2	020A	10 μL ‘P - pSB1A2 – J61100 - ladA – S’	10 μL ‘X – J61107 - ALDH – P’

Emulsifier

Today we tried to see whether P. Putida excreeds an emulsifier. There were 3 samples:

P. Putida grown over night on Glucose (OD: 1.559)
P. Putida grown over night on Octane (OD: 0.838)
M9 medium (negative control) (OD: 0.000)

Therefore we used the following protocol:

Materials:

25 mM triethanolamine (TEA) buffer, pH 8
1% lysozyme in TEA
4M urea
10% streptomycin in TEA
Glass beads
25 mM Tris buffer (pH 8)

Method:

Harvest 25 mL bacterial cells by centrifugation at 10.000 rpm for 10 min
Collect the cell free supernatant and store it
Wash pellet with TEA buffer
Freeze pellet in -70 C for 15 min
Resuspend pellet 2 mL TEA + 40 ul 1% lysozyme (final concentration 0.02%)
Incubate for 10 min at RT
Disrupt cells with glass beads. Vortex for 10 minutes
Centrifuge at 14.000 rpm for 30 min at 4 C (protein is in supernatant)
Aliquot 1.8 mL supernatant into fresh eppendorf tubes and add 200 ul 10% streptomycin in TEA (final concentration 1%)
Incubate 10 min at RT
Centrifuge at 14.000 rpm for 30 min at 4 C (protein is in pellet)
Resuspend pellet in 4 M urea
Centrifuge at 14.000 rpm for 30 min at 4 C (protein is in supernatant)

The samples were tested according to the emulsification assay protocol (@TODO: link):

#	Buffer (ul)	Hexane (ul)	Sample (ul)	Absorbance (660nm)
1	950	50		0.272
2	900	50	50 M9	0.141
3	950	0	50 M9	0.001
4	900	50	50 Glucose culture (sup)	0.100
5	950	0	50 Glucose culture (sup)	0.023
6	900	50	50 Octane culture (sup)	0.161
7	950	0	50 Octane culture (sup)	0.013
8	900	50	50 Glucose culture (pellet)	0.118
9	950	0	50 Glucose culture (pellet)	0.014
10	900	50	50 Octane culture (pellet)	0.168
11	950	0	50 Octane culture (pellet)	- 0.002
12	900	50	50 4 M Urea	0.175
13	950	0	50 4 M Urea	- 0.001

The conclusion is that with the additional of M9 or 4 M Urea the amount of hexane in the Tris buffer decreases. When the samples are used the emulsification becomes even lower. Probably due to the addition of the proteins the total amount of hydrophobic interfaces already increases. Therefore, a lower amount of hexane can be dissolved.

Team:TU Delft/9 August 2010 content