From 2010.igem.org

Lab work

Emulsifier

We have been looking for a robust protocol for the characterization of the emulsifiers for quite some time now. Somehow most literature refers to spectrophotometric assays based on turbidity after mixing. This a somewhat arbitrary method, and so far we were unable to get reproducible results. Today we decided to explore this method once more, using a lot of different conditions.

The method is based on a simple article in Biochemical and Biophysical Methods: Spectrophotometric method for quantitative determination of nonionic, ionic and zwitterionic detergents by Rajakumari et al. 2006.

Calibration graph showing a linear increase in absorbance at 660 nm for increasing concentrations of SDS (●). Under the standard detergent assay conditions, various components like 200 mM NaCl (□), 2.0 mM CaCl2 (♦), 10% glycerol (■), 100 μg microsomal membranes (open diamond), 0.2 mM Triton X-100 (▲) and 2.5 mM CHAPS (○) were added and the turbidity was measured. Error bars represent the deviation from five independent experiments and each one performed in duplicate.

The article shows the graph on the right, which we tried to reproduce. However, instead of triolein we used hexane. And the buffer used was 200 mM Tris-HCl, pH 8.

#	[SDS] (mM)	OD660
1	10	0.598
2	8	0.425
3	6	0.830
4	4	0.432
5	2	0.379
6	0	0.001

This looked promising, so the experiment was carried out once more in triplo and with proper controls.

#	[SDS] (mM)	Serie 1	Serie 2	Serie 3	W/O Hexane
1	8	0.640	0.439	0.622	0.011
2	6	0.454	0.389	0.410	0.014
3	5	0.514	0.539	0.440	0.023
4	4	0.365	0.415	0.449	0.022
5	3	0.346	0.485	0.478	0.028
6	2	0.413	0.404	Sorry, dropped the sample :(	0.024
7	1	0.399	0.500	0.388	0.011
8	0	0.003	0.007	0.004	0.002

As you can see in the Table above, the measurements are kind of random. The way in which you pipet also influences the measurement a lot. That is why we decided to vortex the fluid in the cuvet, and take lower concentrations.

This experiment was also done in triplo:

The conclusion is that hexane dissolves much easier than triolein. This curve might be useful in measuring the emulsification capacity of our proteins. So, next will try to measure the emulsification capacity of our positive control strain P. putida.

Alkane Degradation

Today we're going to try to make the BioBricks again, but using a different method. By cutting the RBSs with SpeI and PstI, so not removing the RBS from the plasmid, and the other piece (gene) with XbaI and PstI, this piece can be inserted into the other plasmid using a 2-way ligation. To prevent the 'insert' plasmid from reclosing and extra cut was done in the plasmid.

Digestion reactions:

#	Fragment	Enzyme 1	Enzyme 2	Enzyme 3 (extra cut)	Used Buffer	BSA
1	rubA3	1μl XbaI	1μl PstI	0.5μl PvuI	3 (Biolabs)	✓
2	rubA4	1μl XbaI	1μl PstI	0.5μl PvuI	3 (Biolabs)	✓
3	rubR	1μl XbaI	1μl PstI	0.5μl EcoRI	3 (Biolabs)	✓
4	ladA	1μl XbaI	1μl PstI	0.5μl PvuI	3 (Biolabs)	✓
5	ADH	1μl XbaI	1μl PstI	0.5μl EcoRI	3 (Biolabs)	✓
6	ALDH	1μl XbaI	1μl PstI	0.5μl PvuI	3 (Biolabs)	✓
7	J61100	1μl SpeI	1μl PstI	-	2 (Biolabs)	✓
8	J61101	1μl SpeI	1μl PstI	-	2 (Biolabs)	✓
7	J61107	1μl SpeI	1μl PstI	-	2 (Biolabs)	✓

The digestion products will be checked on gel tomorrow, but for now the products will be ligated over night:

#	BioBrick	Fragment 1 (recipient vector)	Fragment 2	Final volume
1	K398008A	5 μL J61100	15 μL rubA3	25 μL
2	K398009A	5 μL J61100	9 μL rubA4	20 μL
3	K398010A	5 μL J61100	10 μL rubR	20 μL
4	K398017A	5 μL J61100	12 μL ladA	20 μL
5	K398018A	12 μL J61101	10 μL ADH	30 μL
6	K398019A	13 μL J61107	12 μL ALDH	30 μL

Hydrocarcon Sensing

Because a two-way ligation will be used to place AlkS in front of B0015 and E0422, pSB1A2 containing B0015 and pSB1AK3 containing E0422 will be cut with restriction enzymes EcoRI and XbaI.

Digestion reactions:

#	Sample	Enzyme 1	Enzyme 2	Enzyme 3	Buffer	BSA	Needed fragment
1	1532 μg AlkS	EcoRI	SpeI		Buffer II	✓	‘E – AlkS – S’
2	1532 μg AlkS	EcoRI	SpeI	NcoI	Buffer II	✓
3	822 μg E00422	EcoRI	SpeI	XbaI	Buffer II	✓	‘E X’– E00422
4	1080 μg B0015	EcoRI	XbaI		Buffer II	✓	‘E X’ – B0015

Salt Tolerance

The positive samples from last week were isolated and digested, after which they were used for a two way ligation in an attempt to attach the promoter RBS (BB number J61100).

The digestion was done as follows:

#	content	Volume added (in μl)
1	bbc1-B0015	20 (600 ng)
	Xba	1
	Pst1	1
	BSA	3
	Buffer 2	3
	Water	2
2	J61100	30 (600 ng)
	Spe!	1
	Pst1	1
	BSA	4
	Buffer 2	4

After this step the parts were ligated. (NOTE: this was done in triplicate, once with each of the positive colonies on 23 of july)

Sample	Volume added (in μl)
J61100	5
bbc1-B0015	10
Ligation buffer	2,5
Ligase	0.3 (highly concentrated stock was used)
Water	7.2

Team:TU Delft/26 July 2010 content