Team:NTHU Taiwan/Experiments

Experiments

Experiment - Extracellular Synthesis

In the process of identifying quantum dots synthesis pathway by the bacteria, we predicted two possible pathways: extracellular and intercellular. Since QD synthesis occurs outside the cell for extracellular synthesis, we don't need to break our cell to see the results. For Bacterial culture under Cd medium CdCl ₂ and cystine were added and checked the O.D. value of bacterial culture to reach 0.3. The culture medium itself is the supernatant for the media, which will be observed for color change under UV. For extracellular synthesis in particular the supernatant color will change under UV light.

Figure1. From left to right

Cys0.1mM

1. JM109(cada-yhaO)

2. JM109(cada-yhaO) (Cd ion=10^-6M)

3. JM109(cada-yhaO) (Cd ion=10^-3M)

Cys0.01mM

4. JM109(cada-yhaO)

5. JM109(cada-yhaO) (Cd ion=10^-6M)

6. JM109(cada-yhaO) (Cd ion=10^-3M)

Figure1

As seen above the color changes from left to right for first three and last three tubes respectively. This change supports our hypothesis of cadmium and cystine dependent synthesis of quantum dots by e. coli. Higher concentration of cystine and cadmium means higher concentration of produced quantum dots resulting in sharp color change as seen from figure.

Hence, there will be extracellular synthesis of quantum dots by the bacteria under normal laboratory conditions provided that, cadmium and cystine of right concentration is required.

Experiment - Intercellular synthesis

We attempt to figure it out which pathway does prokaryote E. Coli JM109 take, the intercellular one or the extracellular one. We have the knowledge that if it is intercellular one, the supernatant after centrifuge would be colorless, whereas the extracellular one will show a high saturation color in supernatant.

We use a protocol that added cadmium ion at first, and wash with M9 buffer after centrifuge for twice. With this method, we can make sure all the cadmium that can be used to synthesis quantum dots are all from the inner part of E. Coli. As we can see from the figure, all the tube are under exposure of UV light wavelength = 365nm.

Figure2. The left tube is the one contains E.coli and all the ingredients; the right is only M9 solution. The middle one is the supernatant after centrifuge at 10,000XG for 10mins.

We observed a significant fluorescence emission in the left tube. From the result above we can deduce that, most of the quantum dot is synthesized following intercellular pathway. However, there are still some quantum dots synthesized by the extracellular one by the reason that the supernatant is still emitting a short wavelength blue light under exposure of UV light wavelength = 365nm.

For intercellular synthesis, a step of breaking a cell is required. Only then can the particles be excited by the ultraviolet light. We use lysis buffer and freeze casting method to break the cell. After this procedure, we are able to detect a difference in fluorescence value.

Figure3. The fluorescence value of the lysis result, the supernatant and simple solution.

Figure4. The fluorescence result of the lysis solution, the supernatant, the M9 medium and water under UV ray(365nm).

CadA Experiment

Bacterial strains and plasmids were cultured at 37°C on Luria-Bertani (LB) agar. Standard recombinant DNA techniques were carried out for restriction endonuclease digestion, ligation, transformation of plasmid DNA, and isolation of total DNA

Electrophoresis after digestion of CadA and gene yhaO.

It shows correct location for the CadA(36 b.p). Which when followed by ligation, heat shock and liquid culture shows fluorescence in the range of 450~600nm representing the optimal growth of bacteria and proves that our sample contain cada encoded amplified DNA.

Figure5. Electrophoresis result

Figure6. fluorescence detection

Fluorescence detection of synthesized QD.

Furthermore, we checked the production of quantum dot by our bacteria, for this bacterial growth was done 20mM under different concentration of cystine. Our finding suggests that use of 40μl of cystine with yhaO in the presence of 20μl of cd leads to maximum production of intracellular quantum dots. Maximum fluorescent was observed during three hours time period as per the graph below.

Figure7. The fluorescence value of cada-yhao JM109 (CdCl2: 20mM 20uL)(Cysteine: 20mM 20uL)

Figure8. The fluorescence value of cada-yhao JM109 (CdCl2: 20mM 20uL)(Cysteine: 40mM 20uL)

MntH Experiment

After detect quantum dots fluorescence, we find out that there are two peaks in the detection result which indicate our quantum dots are synthesized through both extracellular and intracellular process. Our dry lab member also use model to calculate the synthesized process, and they suggested our gene design have to solve the problem of different concentration of cadmium .Fortunately, MntH protein can address this problem well since it balances the inside and outside cadmium concentration to a certain degree.

Figure9.

1. coloning

Figure10. MntH cloning result

2. O.D. 600 detection

We want to check the MntH protein is successfully produced by E.coli so we detect the O.D.600 value of two sample, one is the bacteria JM109 contain MntH gene, the other one is the control which only contain wildtype JM109. The result shows that the orange one which contain MntH gene have lower OD value because MntH allows more cadmium ions come into bacteria.

Figure11. O.D. 600 growing curve with cadmium.

Figure12. O.D. 600 growing curve with cadmium 5 hour compare to 10 hour.

MT2 Experiment

1. Cloning

Figure13. MT2 cloning result

2. Growth Curve

In order to test the anti-toxin ability to cadmium,we add the different concentration of cdcl2 to e.coli when it is cultured in LB at 37 degree.The result shows that when the cd concentration range from 10^-9M ~ 10^-5M MT2 can help e.coli against cd.

We also compare the growth curve between wild type JM109 and MT-JM109 .the result show that JM109-MT's growing situation is better than wild type JM109 when the cd concentration isn't too high.

Figure14. anti-cadmium toxin experiment :compare different concentration of cd to JM109-MT

Figure15. anti-cadmium toxin experiment :compare anti-cadmium toxin ability for JM109 to JM109-MT

3. QD test

We were also curious whether MT can help produce QD. So, we use wildtype JM109 and JM109-MT to produce QD. Surprisingly JM109-MT have better emission value on the wavelength range from 400 nm to 500 nm. We propose that the MT can create a steady cadmium location to help sulfur source react with cd well.

Figure16. Fluorescence detection of JM109

Figure17. Fluorescence detection of JM109 -MT

yhaO Experiment

The yhaO gene we used has been specified in E. Coli MG1655[1] that it can use a L-Cysteine desulfhydrases in a pathway that include a redox system that can transform the cysteine into hydrogen sulfur.

In our experiment, we use cysteine and cadmium chloride as our precursor. Cysteine is a relatively common amino acid in biology system. However, it is complex molecule compared to hydrogen sulfide which is the better choice for sulfur resource. The reason that we add cysteine instead of hydrogen sulfide also related to the toxicity of hydrogen sulfide. Still less, hydrogen sulfide exist in gas phase with our experimental environment.

The yhaO gene we used has been specified in E. Coli MG1655 that it can use a L-Cysteine desulfhydrases in a pathway that include a redox system that can transform the cysteine into hydrogen sulfur.

Figure18. The pathway we found in a essay published by Li Kai, Xin Yufeng, Xuan Guanhua, Zhao Rui, Liu Huaiwei, Xia Yongzhen, Xun Luying. Indicating there is two paths to transfer L-cysteine into hydrogen sulfide. We chose CDs(Desulfurhydrase) one after thorough research.

Figure19. We simplified the pathway by reducing the parts we did not opt. This pathway is a essential idea we turn L-cysteine into hydrogen sulfide.

1. Antibiotics test

Figure20. After the precise steps of digestion, ligation, cloning, we are able to see a clear growth on the agar plate which contains C antibiotics. This is one of the ways we verify our cloning result.

As for the relative fluorescence value, we measure the FL spectrum with excitation wavelength = 365 nanometer and emission wavelength=500 nanometer. We can observe a significant change in FL spectrum that both highest intensity and average intensity move toward right. This pattern is matched with those of CdS quantum dots pattern.

2. FL spectrum

Figure21. This figure shows a change in fluorescence(FL) divided by optical density value with time. We can know the relative FL will increase as quantum dots are synthesizing. All the experiment designs here contain cysteine with M9 minimum solution. When cadmium ions are absent(gray line), there’s no FL difference with time changing. Compare the blue curve and orange curve, we can find a proper composition of ingredients.

Figure22.

Reference (yhaO Experiment):

1. Li Kai, Xin Yufeng, Xuan Guanhua, Zhao Rui, Liu Huaiwei, Xia Yongzhen, Xun Luying,Escherichia coli Uses Separate Enzymes to Produce H2S and Reactive Sulfane Sulfur From L-cysteine. Frontiers in Microbiology. VOLUME:10 . 2019 Page 298.
2. Oguri, T., Schneider, B., and Reitzer, L. (2012). Cysteine catabolism and cysteine desulfhydrase (CdsH/STM0458) in Salmonella enterica serovar typhimurium. J. Bacteriol. 194, 4366–4376. doi: 10.1128/JB.00729- 12
3. Functional characterization of a type 2 metallothionein gene, SsMT2, from alkaline-tolerant

Reference (CadA Experiment):

1. Tchounwou P.B., Yedjou C.G., Patlolla A.K., Sutton D.J. (2012) Heavy Metal Toxicity and the Environment. In: Luch A. (eds) Molecular, Clinical and Environmental Toxicology. Experientia Supplementum, vol 101. Springer, Basel.
2. Role of metallothionein in cadmium traffic and toxicity in kidneys and other mammalian organs
3. Functional characterization of a type 2 metallothionein gene, SsMT2, from alkaline-tolerant Suaeda salsa
4. High cadmium-binding ability of a novel Colocasia esculenta metallothionein increases cadmium tolerance in Escherichia coli and tobacco

Reference (MT2 Experiment):

1. Metallothionein: an intracellular protein to protect against cadmium toxicity
2. Role of metallothionein in cadmium traffic and toxicity in kidneys and other mammalian organs
3. Functional characterization of a type 2 metallothionein gene, SsMT2, from alkaline-tolerant Suaeda salsa
4. High cadmium-binding ability of a novel Colocasia esculenta metallothionein increases cadmium tolerance in Escherichia coli and tobacco

CONTACT US

nthuxigem@gmail.com

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