Difference between revisions of "Team:CHINA CD UESTC/Results"

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After our experiments, we successfully constructed the vector piGEM-R-Lac which combined Laccase with RFP by fusion expression. We measured different OD<sub>600</sub> of bacterium at different times, and record the color. We also detected the activity of laccase. We found that the activity of Laccase has positive correlation with OD<sub>600</sub> and the color. We made sure that the activity of Laccase have the catalytic property by using ABTS method. So that we could put them on cathode of EBFC and make it work.
 
After our experiments, we successfully constructed the vector piGEM-R-Lac which combined Laccase with RFP by fusion expression. We measured different OD<sub>600</sub> of bacterium at different times, and record the color. We also detected the activity of laccase. We found that the activity of Laccase has positive correlation with OD<sub>600</sub> and the color. We made sure that the activity of Laccase have the catalytic property by using ABTS method. So that we could put them on cathode of EBFC and make it work.
 
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<strong>(1)SDS-PAGE</strong>
 
<strong>(1)SDS-PAGE</strong>
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<img src="https://static.igem.org/mediawiki/2015/2/2f/CHINA_CD_UESTC_RESULT01.jpg" width="60%">
 
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Lane1: Control. <br>Lane2: Induced expression (BL21).  <br>Induction conditions: 37centigrades，200rpm，induce 10 hours with IPTG which final concentration is 0.5mM.  Using Ultrasonic Cell Disruptor to crush the bacterium in ice-bath.
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Lane1: Control.   Lane2: Induced expression (BL21).  <br>Induction conditions: 37centigrades，200rpm，induce 10 hours with IPTG which final concentration is 0.5mM.  Using Ultrasonic Cell Disruptor to crush the bacterium in ice-bath.
 
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In gel we found that the specific electrophoretic band located near 80KDa, just as we expected.
 
In gel we found that the specific electrophoretic band located near 80KDa, just as we expected.
 
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<strong>(2)Bacterium liquid</strong>
 
<strong>(2)Bacterium liquid</strong>
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<img class="right_pic" src="https://static.igem.org/mediawiki/2015/3/3d/CHINA_CD_UESTC_RESULT03.png" width="380px" height="270px"></div>
 
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<strong>(3)Supernatant after crush：</strong>
 
<strong>(3)Supernatant after crush：</strong>
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we separated it into 3 parts. And we have verified them using digestion (Fig.1) and sequencing.
 
we separated it into 3 parts. And we have verified them using digestion (Fig.1) and sequencing.
 
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We also constructed mamGFDC , mms6, and mamXY which are related to the formation of magnetosome.
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We constructed mamGFDC, mms6, and mamXY which are related to the formation of magnetosome.
 
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<p id="pic_title">The digestion and sequencing of mamW-RFP-Laccase</p>
 
<p id="pic_title">The digestion and sequencing of mamW-RFP-Laccase</p>
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Revision as of 17:18, 10 September 2015

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RESULTS

  We present details on the various methods such as vectors design, domain linker selection and choose of enzyme insertion site that used in the experiment on this page, if you are willing to check or follow our work, you can scan the methods here. Any questions or advice to us are acceptable at any time.

Part 1. Laccase

After our experiments, we successfully constructed the vector piGEM-R-Lac which combined Laccase with RFP by fusion expression. We measured different OD600 of bacterium at different times, and record the color. We also detected the activity of laccase. We found that the activity of Laccase has positive correlation with OD600 and the color. We made sure that the activity of Laccase have the catalytic property by using ABTS method. So that we could put them on cathode of EBFC and make it work.

(1)SDS-PAGE

Lane1: Control. Lane2: Induced expression (BL21).
Induction conditions: 37centigrades，200rpm，induce 10 hours with IPTG which final concentration is 0.5mM. Using Ultrasonic Cell Disruptor to crush the bacterium in ice-bath.

In gel we found that the specific electrophoretic band located near 80KDa, just as we expected.

(2)Bacterium liquid

concentration of bacterium detected at different time

(3)Supernatant after crush：

Using Ultrasonic Cell Disruptor to crush the bacterium in ice-bath. Collect the Supernatant and detect the activity of Laccase by using ABTS. The conclusion is that the activity of Laccasse is increasing with time passing by.

Part2. Vectors construction

we separated it into 3 parts. And we have verified them using digestion (Fig.1) and sequencing.

We constructed mamGFDC, mms6, and mamXY which are related to the formation of magnetosome.

This is the most important vector : mamW+RFP+Laccase.

We have successfully constructed mamAB.

This is part has relation with the formation of magnetosome

The digestion and sequencing of mamW-RFP-Laccase

Part 3. Vectors table

In order to improve the catalytic efficiency of laccase, we decided to immobilize this enzyme. After looking over lots of methods to immobilize laccase. We finally decide to bind laccase to magnetosome. To bind laccase to magnetosome, we designed a series of experiments to let Escherichia coli to express the proteins which are related with the information of the magnetosome, and to get magnetosomes. Through three mouth, we succeed to construct fifteen recombinant plasmids. Details are as follows:

1.For expression

Vector name Inserted gene Gene function
1 piGEM-AB mamAB Encode a series of protein that are essential for magnetosome synthesis
2 piGEM-G6 mamGFDC+mms6 Encode a series of protein that can regulate the size and shape of crystals in the formation of magnetosome
3 piGEM-G6X mamGFDC+mms6+mamXY Encode a series of protein that can regulate the size and shape of crystals in the formation of magnetosome
4 piGEM-R-Lac RFP+laccase Encode a fusion protein which makes laccase visible
5 piGEM-W-R-Lac mamW+RFP+laccase Encode a fusion protein to bind laccase to the transmembrane protein MamW, and RFP can make it visible
6 piGEM-W-Lac mamW+laccase Encode a fusion protein to bind laccase to the transmembrane protein MamW

2.For detection

Vector name Inserted gene Gene function
1 piGEM-Plac-H lac promoter+mamH To make sure whether mamH gene can successfully express
2 piGEM-Plac-G lac promoter+mamG To make sure whether mamG gene can successfully express
3 piGEM-Plac-6 lac promoter+mms6 To make sure whether mamH gene can successfully express
4 piGEM-Plac-Y lac promoter+mamY To make sure whether mamH gene can successfully express
5 piGEM-PH-R-1 mamH promoter+RFP To make sure whether mamH promoter can work
6 piGEM-PG-R mamG promoter+RFP To make sure whether mamG promoter can work
7 piGEM-P6-R mms6 promoter+RFP To make sure whether mms6 promoter can work
8 piGEM-PY-R mamY promoter+RFP To make sure whether mamY promoter can work
9 piGEM-PH-R-2 mamH promoter+RFP To verify the backbone pET28a
10 piGEM-GFP-PG6-R GFP+mamG-mms6 promoter+RFP To make sure whether mamG-mms6 promoter can work