Team:CHINA CD UESTC/Results

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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 OD ₆₀₀ 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 ₆₀₀ 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