Difference between revisions of "Team:Rheda Bielefeld/Assays"

Line 11: Line 11:
 
<div class="column left">
 
<div class="column left">
 
<h2> Pictures: </h2>  
 
<h2> Pictures: </h2>  
−
<img src="https://static.igem.org/mediawiki/2018/4/43/T--Rheda_Bielefeld--Pectin--Assay.png" style="width:100%;height:auto;">  
+
<img src="https://static.igem.org/mediawiki/2018/4/43/T--Rheda_Bielefeld--Pectin--Assay.png" style="width:100%;height:auto;">
 +
<img src="https://static.igem.org/mediawiki/2018/thumb/b/ba/T--Rheda_Bielefeld--Gutes_Pektinbild.jpg/800px-T--Rheda_Bielefeld--Gutes_Pektinbild.jpg" style="width:100%;height:auto;"> 
 +
Results of the assay with assay, left absolute alcohol; right natural alcohol
 +
 
 
<br> <br>
 
<br> <br>
 
The units filled up in eppis, left: 10U; middle: 50U; right: 100U  
 
The units filled up in eppis, left: 10U; middle: 50U; right: 100U  

Revision as of 14:01, 28 November 2018

Home
The Team Our School Bielefeld University Rheda
Description Notebook
Overview Methods Pollen PCR Assays Cloning Strategy
Human Practices Marta Event
Safety Form Biosafety
Attributions Partners

Pictures:

Results of the assay with assay, left absolute alcohol; right natural alcohol

The units filled up in eppis, left: 10U; middle: 50U; right: 100U

Pectin Assay

The so-called chemical pectin is a plant-based polysaccharide which, in terms of nutrition, is a dietary fiber for humans; but some microorganisms are also able to metabolize pectin. To gain pectin, nothing else is necessary as a part of a more genetically complex plant. Even fruits have a relatively high amount of pectin - the shell of citrus fruits reaches peak values of up to 30%. However, the nature of the composition of this pectin depends on the nature of the plant, so pectin from an apple peel is slightly different in construction than that from a lemon peel.

The idea of our project is to help people who are allergic to pollen. Therefore we want to create a biosensor which can detect the amount of different pollen in the air. This provides information about how strongly the allergic person has to dose his or her medicine. To do that, we have to get to the DNA of pollen. For this, however, first the shell of the pollen must be cracked, which also consists of pectin. If we want to get the genetic information out of the pollen, we need a method that can crack pectin. For the pectin assay we have done a series of different experiments to show pectin in chosen substances and to reduce them.

First, we performed a proof for the presence of pectine. Dissolved pectin was mixed with alcohol. As a result, small bubbles had formed in the substance, which was the pectin contained in the substance. Since we were able to successfully detect pectin, we next proceeded to break them down with an enzyme. For this we used the enzyme pectinase from Aspergillus niger and added this to the pectin samples.
The result:
Unlike the previous experiment, no bubbles had formed after the addition of alcohol. The pectin was broken down. These insights are crucial in getting the DNA needed from the pollen and getting a step closer to our ultimate goal.

For observing the reduction of pectin, we tested the samples while we added a certain amount of pectinase enzyme from Aspergillus niger to each pectin sample. In the first container we filled 37µL (10 Units) of enzyme, in the second one 188.5µL (50 Units) and in the third one 337µL (100 Units). We lingered the samples for round about one day and took this observation:
In all of the containers the brown coloration almost completely lessened, caused by the pectinase at the pollen so that the coloration was just hard to see. On the ground, a brown coating settled, probably that was degraded pectin – which we wanted to prove in this experiment. After some time of intensive research, we finally found a suitable protocol with which we could do our pectin assay. For proving pectin, we first dissolved 0.1g of pectin powder (pectin A, pectin C, amylopectin) in 20 ml of distillated water and sequently admitted 5ml of pure ethanol and denatured ethanol. For the negative control, we filled 5ml of water in three test tubes and mixed it with 2.5ml of the pectin samples.

We also tried to implement this assay with apple and orange juice because the peels of fruits consist of a lot of pectin. After a while we noticed little bubbles which formed in the substances and were mixed with ethanol. We were able to see those small bubbles as well in the pectin samples as in the juice samples. That meant that we were able to prove pectin in different substances. After that we tried to prove the destruction of pectin via pectinase. Therefore we filled the substances in the eppis in three test tubes each and added pure ethanol. Incidentally, we prepared 4 control samples, one contained 1 ml of pure pectin A, in the other 3 we filled each for the purpose of negative control, the respective volumes of pectinase added in the last experiment in the form of water. The result:
While we could see bubbles in the pectin proving test, the positive control substance was liquid and cloudy, but did not contain bubbles.
Therefore, it is assumed that we could reduce pectin due to the enzyme. The positive test had worked.

Although the positive control worked very well, there is still place for improvements. Soon, it could get found out if we could use less units so that we can use them for more tries. In addition, it should be checked whether the assay would work for other sorts of pectin as well.

Cellulose Assay

Our pectin assay is actually associated with a related cellulose assay.
The complexity of miscellaneous methods and the conditions of time however made trouble with finishing our experiments, so that we can just explain what our plans and thoughts theoretically had been on this topic.

Cellulose makes up 50% of the construction of cell walls, which makes cellulose the basic component of those. Like any other plant cell in the nature, also the cores of pollen have cell walls made of cellulose, so our goal is to break through the wall of the pollen. This is a necessary step for getting the DNA of the pollen.

In this case our cellulose assay goes with the measurement of the viscosity in endoglucanase while we put the cellulose hydroethylcellulose within. Endoglucanase tends to destruct chemical bonds.
The advantage now is that it is possible to measure up the viscosity of cellulose. Therefore you need to find the current rate of change by mathematic methods of calculation. This gives information about how good destruction of cellulose is running.

Sources: