Team:TU Delft/26 July 2010 content

From 2010.igem.org

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=Emulsification Assay=
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==Emulsification Assay==
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.
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.
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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''.
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''.
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 +
==Alkane Degradation==
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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.
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[[Team:TU_Delft/protocols/restriction_enzyme_digestion_40| Digestion reactions]]:
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|'''#'''
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|'''Fragment'''
 +
|'''Enzyme 1'''
 +
|'''Enzyme 2'''
 +
|'''Enzyme 3 (extra cut)'''
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|'''Used Buffer'''
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|'''BSA'''
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|-
 +
|1
 +
|rubA3
 +
|XbaI
 +
|PstI
 +
|PvuI
 +
|3 (Biolabs)
 +
|✓
 +
|-
 +
|2
 +
|rubA4
 +
|XbaI
 +
|PstI
 +
|PvuI
 +
|3 (Biolabs)
 +
|✓
 +
|-
 +
|3
 +
|rubR
 +
|XbaI
 +
|PstI
 +
|EcoRI
 +
|3 (Biolabs)
 +
|✓
 +
|-
 +
|4
 +
|ladA
 +
|XbaI
 +
|PstI
 +
|PvuI
 +
|3 (Biolabs)
 +
|✓
 +
|-
 +
|5
 +
|ADH
 +
|XbaI
 +
|PstI
 +
|EcoRI
 +
|3 (Biolabs)
 +
|✓
 +
|-
 +
|6
 +
|ALDH
 +
|XbaI
 +
|PstI
 +
|PvuI
 +
|3 (Biolabs)
 +
|✓
 +
|-
 +
|7
 +
|J61100
 +
|SpeI
 +
|PstI
 +
| -
 +
|2 (Biolabs)
 +
|✓
 +
|-
 +
|8
 +
|J61101
 +
|SpeI
 +
|PstI
 +
| -
 +
|2 (Biolabs)
 +
|✓
 +
|-
 +
|7
 +
|J61107
 +
|SpeI
 +
|PstI
 +
| -
 +
|2 (Biolabs)
 +
|✓

Revision as of 14:08, 27 July 2010

Emulsification Assay

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) Absorbance 660 nm (AU)
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 XbaI PstI PvuI 3 (Biolabs) ✓
2 rubA4 XbaI PstI PvuI 3 (Biolabs) ✓
3 rubR XbaI PstI EcoRI 3 (Biolabs) ✓
4 ladA XbaI PstI PvuI 3 (Biolabs) ✓
5 ADH XbaI PstI EcoRI 3 (Biolabs) ✓
6 ALDH XbaI PstI PvuI 3 (Biolabs) ✓
7 J61100 SpeI PstI - 2 (Biolabs) ✓
8 J61101 SpeI PstI - 2 (Biolabs) ✓
7 J61107 SpeI PstI - 2 (Biolabs) ✓
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