Team:TU Delft/26 July 2010 content

From 2010.igem.org

(Difference between revisions)
(→Salt Tolerance)
(→Salt Tolerance)
Line 303: Line 303:
|4
|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)
 +
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''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
 +
|-
|}
|}

Revision as of 11:33, 10 August 2010

Contents

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

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
Retrieved from "http://2010.igem.org/Team:TU_Delft/26_July_2010_content"