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

Revision as of 02:10, 14 September 2015

<!DOCTYPE html>

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.

Part1.

We separated mamAB into 3 parts and amplified each one by PCR.

Figure 1.1: We amplified three parts of mamAB by PCR using high fidelity polymerase Advantage.

We used three parts to constructed mamAB successfully by subclone and we have verified them using digestion (Fig.1) and sequencing.

Figure 1.2: We confirmed mamAB by enzyme digestion using ApaI、SapI、NotI enzymes

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

Figure 1.3: We amplified mamGFDC+mms6 and mamXY by PCR using high fidelity polymerase Advantage.

This is part has relation with the formation of magnetosome

Figure 1.4: We confirmed piGEM-G6X by enzyme digestion using Hind III enzymes in Lane1 and Apal I and Pst I enzymes in Lane2.

We respectively amplified three fragments by PCR.

Figure 1.5: We amplified mamW and RFP and Laccase by PCR using high fidelity polymerase Advantage.

We successfully connected three fragments together by fusion PCR

Figure 1.6: We amplified mamW+RFP+Laccase and RFP+ Laccase by fusion PCR using high fidelity polymerase Advantage.

We recovered the PCR products and succeddfully inserted the fragment into pACYCDuet. and confirmed piGEM-WRL and piGEM-RL by restriction digestion.

Figure 1.7: We confirmed piGEM-WRL by enzyme digestion using Pst1 +Xho1 enzymes

Figure 1.8: We confirmed piGEM-RL by enzyme digestion using Pst1 +Xho1 enzymes

Part 2.

After our experiments, we successfully combine mamW,Laccase and RFP. We measured different OD of bacterium at different time, and record the color. We also detected the activity of Laccase. We found that the activity of Laccase has positive correlation with OD and the color. We make sure that the activity of Laccase have the catalytic property by using ABTS method. So that we can put them at cathode of EBFC and make it work.

(1)SDS-PAGE

Lane1 :control of pACYCDuet-1-RFP-Laccasse
Lane2 :BL21 Bacterial strain which contain pACYCDuet-1-RFP-Laccasse induced expression.

Induction fonditions:37centigrades，200rpm，induce 10 hours with IPTG which final concentration is 0.5mM. Using Ultrasonic Cell Disruptor to crush the bacterium in ice-bath.

Add 6*SDS-PAGE loading buffer，put it in boiling water bath for 5minius . The size of the target protein is 84KDa which is fusion expressed by RFP+Laccse,In gel we found that the line Bacterial strain which contain pACYCDuet-1-RFP-Laccasse is located near 80KDa, just as we expected.

(2)Bacterium liquid

concentration of bacterium detected at different time

Figure 2.2: We compared color degree among different bacterium liquid. The higher concentration of bacterium showed redder.

Figure 2.3: We measured the OD of different concentration of baterium at different time using ultraviolet spectrophotometer

(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 as the time pass the Laccasse is more.

Figure 2.4: We broke the bacterium at different time and extracted the supernate and compared color degree among different bacterium liquid. The higher concentration of bacterium showed redder.

Figure 2.5: We determined enzymes activities from different supernate using ABTS method. Every supernate presents the different bacterium which are cultivated for different time.

mamW+RFP+Laccase

We transferred piGEM-WRL into BL21 bacterial strain and got a series of bacter-ium liquid which are cultivated for different time.

Bacterium liquid

We compared one of the experimental groups with control group and the experimental group showed red color which indicated the this expressed successfully. But the color is dim compared with the enzyme coded by piGEM-RL which indicated the enzyme coded by piGEM-WRL activity is weaker.

Figure 2.6: We choose one to compared with BL21 bacterial strain containing empty vector as control group and showed that experimental group is redder than control group.

We broke the bacterium liquid and extracted the supernate. Then we measured the activities of enzyme from different supernate with the same method.

Figure 2.7: We determined enzymes activities from different supernate using ABTS method. Every supernate presents the different bacterium which are cultivated for different time.

In the end, we compared the two enzyme activity curves. The one represented the enzyme coded by piGEM-RL, the other is coded by piGEM-WRL. This picture showed that the enzyme activity of the laccase coded by piGEM-RL is higher than the other.

Figure 2.8: two enzyme activity curves presented the two different laccases coded by different vector. the red curve represented piGEM-RL and the blue curve presented piGEM-WRL.

Part 3.

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

Part 4.

Device 1：EBFC 1.0

Figure 4.1. Make this device using discarded bottles with the proton exchange membrane in the middle.

Figure 4.2. Fix the device 1 in the foam board, add each component (10 ml in total) according to the sketch. Use multimeter and oscilloscope to test voltage.

Due to the fact that the sealing was not very good and there existed leakage phenomenon, after several tests, the voltage is basically 0. After a resistance measure by the multimeter, we found that the battery’s internal resistance is too high, so we need to further improve the device.

Device 2: EBFC 2.0

Figure 4.3. The middle of the device is proton exchange membrane; the electrode material is carbon paper. Run the device.

Figure 4.4. Before adding enzyme, the voltage is 0; the right is the scan map of oscilloscope in the left.

After adding enzyme:

Figure 4.5. the voltage increased instantly the moment when adding enzyme, but the voltage was far from stable. After about 5 minutes, the voltage gets stable.

Figure 4.7. the voltage reached a stable level of 155mV. The scan time is 50s and every grid represents 100mV.

From the upper picture, we can see that the EBFC effected the desired result—produced voltage, the highest voltage reached 0.25V and lasted for____. But the battery’s internal resistance was too large, resulting in the small electric current, so we need to further improve the battery.

Device 3: EFBC 3.0

Figure 4.8. 3D - print EBFC device.

Run the 3D print device and test, it didn’t achieve the desired result; the internal resistance of the battery didn't get smaller. In order to further improve the energy-producing effect and make enzyme catalyze substrates constantly, we need to find a better method to make enzyme gathered on the surface of two electrodes and fixed, so enzyme wouldn’t decrease with the change of substrates, realizing enzyme’s constant catalysis and improving the longevity of the battery.

Results of the promoter validation work:

In order to find the reason why magnetosomes canot form, we designed experiments to validate the promoters of 4 operons (connected to the design page).We successfully constructed the promoter validation work related vectors and transferred them to E. coli, cultured at 37 ℃and 180 RPM for 24 hours, but the bacteria liquid didn’t turn red. Then we broke cells and operate SDS-page electrophoresis, but we also didn’t find an RFP corresponding electrophoretic band. It meant that the native promoters related to the formation of magnetosomes in magnetotactic bacteria can’t work in E.coli, so finally we didn’t get magnetosomes.

@@ Line 570: / Line 570: @@
 									<img src="https://static.igem.org/mediawiki/2015/4/49/CHINA_CD_UESTC_RESULT22.png" width="340px" height="240px">
 									<p id="pic_illustration">
-									Figure 4.7. the voltage reached a stable level of 155mV. The scan time is 50s and every grid represents 100Mv.
+									Figure 4.7. the voltage reached a stable level of 155mV. The scan time is 50s and every grid represents 100mV.
 									</p>
 								</div>
 							</div>
 							<p>
-								from the upper picture, we can see that the EBFC effected the desired result—produced voltage, the highest voltage reached 0.25V and lasted for____. But the battery’s internal resistance was too large, resulting in the small electric current, so we need to further improve the battery.
+								From the upper picture, we can see that the EBFC effected the desired result—produced voltage, the highest voltage reached 0.25V and lasted for____. But the battery’s internal resistance was too large, resulting in the small electric current, so we need to further improve the battery.
 							</p>
 							<p>
-								<strong>EFBC 3.0</strong>
+								<strong>Device 3: EFBC 3.0</strong>
 							</p>
 							<div class="project_pic">