Difference between revisions of "Part:BBa K2242005"
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KMADH is an alcohol dehydrogenase from Kluyveromyces marxianus. It can catalyze the ethanol fermentation ongoing in the bacteria with a high efficiency and mild reaction condition, especially temperature.
 
KMADH is an alcohol dehydrogenase from Kluyveromyces marxianus. It can catalyze the ethanol fermentation ongoing in the bacteria with a high efficiency and mild reaction condition, especially temperature.
 
We use T7 promoter and Lac operator to control KMADH’s expression. Both of this two units are induced by IPTG. With this dual switch, we can reduce the leak of the gene expression as much as possible.
 
We use T7 promoter and Lac operator to control KMADH’s expression. Both of this two units are induced by IPTG. With this dual switch, we can reduce the leak of the gene expression as much as possible.
 +
 +
    In our project, Mtr CAB is the most important and fundamental proteins as it plays the role to transfer extracellular electrons into the cytoplasm through the membrane. To examine whether the Mtr protein complex has the function as we expected, we used our engineered strain pMC( strain co-expressed Mtr CAB and Ccm A-H) to construct a bio-cathode. We monitored the current of the bio-cathode to see whether there would be a higher current in the experiment group than the WT strain.
 +
    Here, first we did an bacteria PCR to monitor the maintenance of the recombinant plasmids(pM28 contains the mtr CAB’s gene and the pTBC contains the ccm A-H’s gene). As we can see in figure 11, we could confirm that the strain is fine to use.
 +
    https://static.igem.org/mediawiki/2017/9/94/USTC-result-Mtr-1.png
 +
    Figure 11. Electrophoresis result of PCR of Mtr and Ccm
 +
    So we started our bio-cathode assay to examine our theory. The protocol of the bio-cathode assay can be found in the notebook part in our wiki, look it up if you want to know more details!! In figure 12, we can easily confirm that the Mtr CAB protein complex was mature, as the pellets were red in pMC group, no matter the strain had been induced or not. When we did it the first time, there was no significant difference of the current of the bio-cathode between WT and our strain pMC(data not shown). We speculated that it was because we did NOT have the starvation step when we first did it, which is to cultivate the bacteria in a minimal salts medium for a certain time, like 4 to 6 hours. Because we did NOT have this starvation step, although we already used PBS to wash the bacteria 2 to 3 times, those nutritions still contained inside of the bacteria, providing another electron source when we were running the bio-cathode. So when the cathode was given a certain voltage, the bacteria still wouldn’t take up the electrons from the electrode.
 +
    https://static.igem.org/mediawiki/2017/6/6f/USTC-result-Mtr-2.jpeg
 +
Figure 12. Bacteria sediments<br>(from left to right, WT, pMC not induced, pMC induced)
  
 
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    In our project, Mtr CAB is the most important and fundamental proteins as it plays the role to transfer extracellular electrons into the cytoplasm through the membrane. To examine whether the Mtr protein complex has the function as we expected, we used our engineered strain pMC( strain co-expressed Mtr CAB and Ccm A-H) to construct a bio-cathode. We monitored the current of the bio-cathode to see whether there would be a higher current in the experiment group than the WT strain.</p>
+
 
−
    Here, first we did an bacteria PCR to monitor the maintenance of the recombinant plasmids(pM28 contains the mtr CAB’s gene and the pTBC contains the ccm A-H’s gene). As we can see in figure 11, we could confirm that the strain is fine to use. </p>
+
 
−
    https://static.igem.org/mediawiki/2017/9/94/USTC-result-Mtr-1.png
+
−
    Figure 11. Electrophoresis result of PCR of Mtr and Ccm
+
−
    So we started our bio-cathode assay to examine our theory. The protocol of the bio-cathode assay can be found in the notebook part in our wiki, look it up if you want to know more details!! In figure 12, we can easily confirm that the Mtr CAB protein complex was mature, as the pellets were red in pMC group, no matter the strain had been induced or not. When we did it the first time, there was no significant difference of the current of the bio-cathode between WT and our strain pMC(data not shown). We speculated that it was because we did NOT have the starvation step when we first did it, which is to cultivate the bacteria in a minimal salts medium for a certain time, like 4 to 6 hours. Because we did NOT have this starvation step, although we already used PBS to wash the bacteria 2 to 3 times, those nutritions still contained inside of the bacteria, providing another electron source when we were running the bio-cathode. So when the cathode was given a certain voltage, the bacteria still wouldn’t take up the electrons from the electrode.</p>
+
−
        <img src="https://static.igem.org/mediawiki/2017/6/6f/USTC-result-Mtr-2.jpeg" width="40%" style="margin:0 30%;">
+
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        <p style="text-align:center!important">Figure 12. Bacteria sediments<br>(from left to right, WT, pMC not induced, pMC induced)</p>
+
 
        <p class="indent_word">So we performed this bio-cathode assay for a second time, adding this starvation step into the protocol. In addition, after the starvation step, we used 1 mL of minimal salts medium to resuspend the bacteria and dropped it onto the graphite electrode to form a bio-film, which could help to make a better connection between the bacteria and the electrode, especially when we were using the Mtr pathway to transfer electrons. Here, in figure 13, you can see how we made this biofilm. 2 to 3 hours later, with a sufficient airflow in the laminar flow hood, the graphite electrode would dry up and form a great biofilm. With this biofilm, electrons could be transferred to the Mtr C protein directly from the electrode which can increase the efficiency of electron transferring. Then what we need to do was to construct this bio-cathode, put every part of this “toy” together and get the oxygen out of this container. Here in figure 14 is how we clear the oxygen out of the bio-cathode to create an anaerobic environment. Lastly, we connected the bio-cathode to the electric-chemical station to give a certain voltage to the cathode and monitor the current of the cathode as time went by as how figure 15 shows. </p>
 
        <p class="indent_word">So we performed this bio-cathode assay for a second time, adding this starvation step into the protocol. In addition, after the starvation step, we used 1 mL of minimal salts medium to resuspend the bacteria and dropped it onto the graphite electrode to form a bio-film, which could help to make a better connection between the bacteria and the electrode, especially when we were using the Mtr pathway to transfer electrons. Here, in figure 13, you can see how we made this biofilm. 2 to 3 hours later, with a sufficient airflow in the laminar flow hood, the graphite electrode would dry up and form a great biofilm. With this biofilm, electrons could be transferred to the Mtr C protein directly from the electrode which can increase the efficiency of electron transferring. Then what we need to do was to construct this bio-cathode, put every part of this “toy” together and get the oxygen out of this container. Here in figure 14 is how we clear the oxygen out of the bio-cathode to create an anaerobic environment. Lastly, we connected the bio-cathode to the electric-chemical station to give a certain voltage to the cathode and monitor the current of the cathode as time went by as how figure 15 shows. </p>
 
        <div class="col s6">
 
        <div class="col s6">

Revision as of 20:24, 27 October 2017


T7+lacO+KmAdh

KMADH is an alcohol dehydrogenase from Kluyveromyces marxianus. It can catalyze the ethanol fermentation ongoing in the bacteria with a high efficiency and mild reaction condition, especially temperature. We use T7 promoter and Lac operator to control KMADH’s expression. Both of this two units are induced by IPTG. With this dual switch, we can reduce the leak of the gene expression as much as possible. 

   In our project, Mtr CAB is the most important and fundamental proteins as it plays the role to transfer extracellular electrons into the cytoplasm through the membrane. To examine whether the Mtr protein complex has the function as we expected, we used our engineered strain pMC( strain co-expressed Mtr CAB and Ccm A-H) to construct a bio-cathode. We monitored the current of the bio-cathode to see whether there would be a higher current in the experiment group than the WT strain.
   Here, first we did an bacteria PCR to monitor the maintenance of the recombinant plasmids(pM28 contains the mtr CAB’s gene and the pTBC contains the ccm A-H’s gene). As we can see in figure 11, we could confirm that the strain is fine to use. 
   USTC-result-Mtr-1.png
   Figure 11. Electrophoresis result of PCR of Mtr and Ccm
   So we started our bio-cathode assay to examine our theory. The protocol of the bio-cathode assay can be found in the notebook part in our wiki, look it up if you want to know more details!! In figure 12, we can easily confirm that the Mtr CAB protein complex was mature, as the pellets were red in pMC group, no matter the strain had been induced or not. When we did it the first time, there was no significant difference of the current of the bio-cathode between WT and our strain pMC(data not shown). We speculated that it was because we did NOT have the starvation step when we first did it, which is to cultivate the bacteria in a minimal salts medium for a certain time, like 4 to 6 hours. Because we did NOT have this starvation step, although we already used PBS to wash the bacteria 2 to 3 times, those nutritions still contained inside of the bacteria, providing another electron source when we were running the bio-cathode. So when the cathode was given a certain voltage, the bacteria still wouldn’t take up the electrons from the electrode.
   USTC-result-Mtr-2.jpeg

Figure 12. Bacteria sediments
(from left to right, WT, pMC not induced, pMC induced)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 860
    Illegal XhoI site found at 922
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]




So we performed this bio-cathode assay for a second time, adding this starvation step into the protocol. In addition, after the starvation step, we used 1 mL of minimal salts medium to resuspend the bacteria and dropped it onto the graphite electrode to form a bio-film, which could help to make a better connection between the bacteria and the electrode, especially when we were using the Mtr pathway to transfer electrons. Here, in figure 13, you can see how we made this biofilm. 2 to 3 hours later, with a sufficient airflow in the laminar flow hood, the graphite electrode would dry up and form a great biofilm. With this biofilm, electrons could be transferred to the Mtr C protein directly from the electrode which can increase the efficiency of electron transferring. Then what we need to do was to construct this bio-cathode, put every part of this “toy” together and get the oxygen out of this container. Here in figure 14 is how we clear the oxygen out of the bio-cathode to create an anaerobic environment. Lastly, we connected the bio-cathode to the electric-chemical station to give a certain voltage to the cathode and monitor the current of the cathode as time went by as how figure 15 shows. 

                                                       <img src="800px-USTC-demo-3.jpeg" width="80%" style="margin:0 4%;">

Figure 13. Preparation for bio-film

                                                       <img src="800px-USTC-demo-2.jpeg" width="80%" style="margin:0 4%;">

Figure 14. Preparation for reaction system
(to exclude oxygen out of the container)

<img src="800px-USTC-result-Mtr-5.jpeg" width="40%" style="margin:0 30%;">

Figure 15. Bio-cathode device

Figure 16 is the result of this experiment. From the figure, we can easily notice that the red line, which is the Mtr-induced group, had a 50% higher current than the other two group after the bio-cathode turned into a stable state . This could strongly prove that the engineered strain pMC can transfer electrons into the cytoplasm, which led to the increasing of the cathode-current. But there would be a chance that this difference between these 3 groups was just the background noise between this three cathode, resulting from the hardware’s varieties. So we added fumarate into the system to see whether there would be a cathode catalyzed current happened in the pMC group. That’s why there was a sharp increasing in the figure. When we added fumarate into the system, the electrons on the electrode finally found a way to leak to—— the fumarate. So there would be a strong electron flow when we added fumarate into the system. But after a short time we introduced this sudden change into the system, the current will become stable again, slowly climbing back to the current it was. However, the time it took to get back to stable state can be a strong evident to prove our assumption——our engineered E.coli can transfer extracellular electrons into the cytoplasm!! The red line’s curve happened after we added fumarate into the system is kind of a typical curve of cathode-catalyze-current!! So, with this result, the cathode’s current to time under a certain voltage, we can confidently say that the Mtr CAB system work!!

<img src="USTC-result-Mtr-10.jpeg" width="80%" style="margin:0 10%;">

Figure 16. The current result of the bio-cathode.

In conclusion, the Mtr CAB system can really function as an electron pathway to transfer extracellular electrons into the cytoplasm, even though it’s expressed in E.coli, but not it’s origin host Shewanella.!! In another word, our conduction system can function as we expected, transferring those electrons from the electrode into the cytoplasm, which means our E.coli can transform itself like transformer from a normal form to a special form that can “eat” electrons!


Reference:

[1] Thomas, P. E., Ryan, D., & Levin, W. (1976). An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Analytical biochemistry, 75(1), 168-176.
[2] Jensen, H. M. (2013). Engineering Escherichia coli for molecularly defined electron transfer to metal oxides and electrodes. University of California, Berkeley </div>