Team:BNDS China/Contribution
Contribution
Overview
In synthetic biology, we always need to measure the concentration of a kind of substance in solution to check the yield of the product. There are several maturely developed methods to do that, such as HPLC, which can give out detailed and accurate concentration of a kind of molecule. However, it is complicated and difficult to operate, and the equipment is hard for students to get in touch with. In that case, we want to find an approach which can give out result fast and specific for our topic, rhamnolipid test. Therefore, we introduce oil spreading as an easy way to qualitatively compare the concentration of lipid or anything that are hydrophobic.
Protocol
1.Add Sudan red into paraffin until it is saturated.
2.Add 30 ml water into a clean, sterilized medium. 3.Use a pipette gun to spread 8 ml saturated Sudan red paraffin on the center of surface of water, each time spread 1000 ul of paraffin each time, and don’t let paraffin sink into water.
4.Since paraffin tend to accumulate near the wall of medium, pipette gun is used to extract those paraffin and add it on the center of medium.
2.Add 30 ml water into a clean, sterilized medium. 3.Use a pipette gun to spread 8 ml saturated Sudan red paraffin on the center of surface of water, each time spread 1000 ul of paraffin each time, and don’t let paraffin sink into water.
4.Since paraffin tend to accumulate near the wall of medium, pipette gun is used to extract those paraffin and add it on the center of medium.
Notice
Remember to spread oil on water, and if there are any oil drops sink beneath the surface, use pipette gun to take it out and spread on surface again.
figure.1 Ensure paraffin layer is floating on water
Using oil spreading, our team can measure the concentration of rhamnolipid in bacteria liquid easily. The picture beneath is one of the result of rhamnolipid testing. By measuring the diameter of oil circle, we got an approximate result of concentration of rhamolipid, which allows us to check the yield of enzyme in E.coli after we transplant plasmid containing rhl A, rhl B, and rhl C into it.
Figure.2 A oil circle that expands
Our Result
We add different concentration of rhamnolipid to do oil spreading test, from 100 ug/L to 900ug/L, to prove that oil spreading is a reliable method to test concentration of rhanolipid.
Figure.3 shows the result. The r2 value larger than 0.8 so that it have significance in statistic, and prove the validity of oil spreading as a reliable method in measuring concentration.
When the concentration increase, the rate of increase of diameter decrease. We speculate that it is caused by the increase of repulsion force. With larger diameter, the oil need to push larger volume of water. The reason why the diameter reaches a plateau is that the diameter of our culture dish is about 7 cm.
Figure.3 Graph with concentration on x axis and diameter on y axis and a linear trendline.
Conclusion
We propose that oil spreading, a very simple, cheap, and effective way to qualitatively measure the concentration of oil like substances, as a convenient and accessible test method for high school students to operate. Even though compared with HPLC, it may lack quantitative result, it is still enough for us to check the concentration of hydrophobic substances.