Difference between revisions of "Team:TCU Taiwan/Experiments"

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<p>Describe the experiments, research and protocols you used in your iGEM project.</p>
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<h5>What should this page contain?</h5>
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<ul>
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<li> Protocols </li>
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<li> Experiments </li>
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<li>Documentation of the development of your project </li>
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<h5>Inspiration</h5>
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<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
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<td align=center><font size=25>Experiments<br><br></font></td>
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<th align=left><span style="font-family:Calibri;text-align:justify;"><font size="5">
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In order to produce the CHROMO DIABETECTOR, we need to perform some experiments. To complete this product, we should get the sequence of Red color chromo-protein and <I>ptsG</I>. After we got the sequences for both the Red color chromo-protein and <I>ptsG</I>, we need to perform the first restriction and ligation to our plasmid and then transform them into the <I>E. coli</I>.<br><br>
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Here’s the experiments that we did to create Chromo Diabetector:<br><br>
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Red color chromo protein:<br><br>
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1. Making the competent cells: To enable the <I>E. coli</I>  to uptake extracellular genetic material (red color chromo protein), we need to make the membrane of it to be permeable for our red color chromo protein.

<br><br>
 +
2. Transformation: After the <I>E. coli</I> has become a competent cell, now we can transform our red color chromo protein <I>(BBa_k592012)</I> which already exists in the <I>PSB1C3</I> plasmid into the competent cell.

<br><br>
 +
3. Recombinant plasmid culturing: Now we can culture the <I>E. coli</I> that with our red color chromo protein in the LB agar media by adding the selective marker and the plasmid that we have already transform into the <I>E. coli</I>.

<br><br>
 +
4. Plasmid extraction from <I>E. coli</I>: Pick out one of the colony which grow in our agar plate and put it into the modified LB media to growth in the incubator for overnight then we can start to extract the plasmid out.<br><br>
 +


5. Measuring the concentration: Measured the extracted plasmid value, when the value reaches a certain ranges then we can continue over to the PCR.

<br><br>
 +
6. PCR: Using the designed primer, we can take the red color chromo protein sequence out from our plasmid.

<br><br>
 +
7. Enzyme cutting: We used the restriction enzymes to cut down our designed site. After it precipitated, we can go through the next step.<br><br>
 +

8. Gel electrophoresis: Run the gel electrophoresis to check whether we get the right sequence of the red color chromo protein.

<br><br>
 +
9. Gel purification: Perform the Gel purification of the gel electrophoresis, to get the purified red color chromo protein from the gel.

<br><br>
 +
10. Get the sequence: Save the red color chromo protein sequence in the -20 fridge for the long term usage.
<br><br>
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<I>ptsG</I>:

<br><br>
 +
1. DNA isolation from <I>E. coli</I>: Because the sequence of <I>ptsG</I> is already exist in the <I>E. coli BL21</I>, what we need to do is to extract the DNA from the <I>E. coli</I>.

<br><br>
 +
2. Measuring the concentration: After we extract the DNA from the <I>E. coli</I>, we should make sure the concentration of the DNA is suitable for the PCR.

<br><br>
 +
3. PCR: By designing the primer for it, we can get the <I>ptsG</I> sequence.

<br><br>
 +
4. PCR purification: Perform the PCR purification to isolate the <I>ptsG</I> sequence from the PCR material.

<br><br>
 +
5. Gel electrophoresis: This is to ensure that we got the correct sequence of the <I>ptsG</I>.<br><br>
 +


6. Gel purification: Perform the Gel purification to isolate the <I>ptsG</I> sequence from the Gel electrophoresis material.
<br><br>
 +
Final step:

<br><br>
 +
When we have both the <I>ptsG</I> sequence and the sequence of the red color chromo protein, the next step is to put them inside the plasmid backbone by using restriction enzyme and the ligation enzyme.

<br><br>
 +
1. Restriction enzyme cutting <I>PQE60</I> : Cut the <I>PQE60</I> plasmid, <I>ptsG</I> sequence and the red color chromo protein sequence by using the restriction enzymes.

<br><br>
 +
2. Ligase red and <I>ptsG</I> sequences in <I>PQE60</I>: Ligase both the red chromo protein sequence and the <I>ptsG</I> sequence by using the ligation enzyme.

<br><br>
 +
3. Transform recombinant plasmid into <I>dh5α</I>: Transforming our recombinant plasmid into the <I>dh5α</I> is one of the method to confirm whether or not we are successful in the ligation process. If there is any colonies when we are culturing the <I>dh5α</I>, then we can transform our recombinant plasmid into <I>E. coli BL21</I>. The purpose of choosing <I>dh5α</I> is because the <I>dh5α</I> does not contain any plasmids. Hence, enabling us to confirm whether or not we performed the correctly ones in the ligation phase.<br><br>
 +
4. Transform recombinant plasmid into <I>E. coli BL21</I>: The reason we choose the <I>E. coli BL21</I> because it can tolerate in high glucose concentration.<br><br>
 +
After we have done all of the step above, we are able to produce the Chromo Diabetector. Although the steps look easy, but it requires multiple test and tries to get the correct sequence of both the <I>ptsG</I> and the red color chromo protein.
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Latest revision as of 15:55, 19 October 2016

Experiments
In order to produce the CHROMO DIABETECTOR, we need to perform some experiments. To complete this product, we should get the sequence of Red color chromo-protein and ptsG. After we got the sequences for both the Red color chromo-protein and ptsG, we need to perform the first restriction and ligation to our plasmid and then transform them into the E. coli.

Here’s the experiments that we did to create Chromo Diabetector:

Red color chromo protein:

1. Making the competent cells: To enable the E. coli  to uptake extracellular genetic material (red color chromo protein), we need to make the membrane of it to be permeable for our red color chromo protein.



2. Transformation: After the E. coli has become a competent cell, now we can transform our red color chromo protein (BBa_k592012) which already exists in the PSB1C3 plasmid into the competent cell.



3. Recombinant plasmid culturing: Now we can culture the E. coli that with our red color chromo protein in the LB agar media by adding the selective marker and the plasmid that we have already transform into the E. coli.



4. Plasmid extraction from E. coli: Pick out one of the colony which grow in our agar plate and put it into the modified LB media to growth in the incubator for overnight then we can start to extract the plasmid out.



5. Measuring the concentration: Measured the extracted plasmid value, when the value reaches a certain ranges then we can continue over to the PCR.



6. PCR: Using the designed primer, we can take the red color chromo protein sequence out from our plasmid.



7. Enzyme cutting: We used the restriction enzymes to cut down our designed site. After it precipitated, we can go through the next step.


8. Gel electrophoresis: Run the gel electrophoresis to check whether we get the right sequence of the red color chromo protein.



9. Gel purification: Perform the Gel purification of the gel electrophoresis, to get the purified red color chromo protein from the gel.



10. Get the sequence: Save the red color chromo protein sequence in the -20 fridge for the long term usage.


ptsG:



1. DNA isolation from E. coli: Because the sequence of ptsG is already exist in the E. coli BL21, what we need to do is to extract the DNA from the E. coli.



2. Measuring the concentration: After we extract the DNA from the E. coli, we should make sure the concentration of the DNA is suitable for the PCR.



3. PCR: By designing the primer for it, we can get the ptsG sequence.



4. PCR purification: Perform the PCR purification to isolate the ptsG sequence from the PCR material.



5. Gel electrophoresis: This is to ensure that we got the correct sequence of the ptsG.



6. Gel purification: Perform the Gel purification to isolate the ptsG sequence from the Gel electrophoresis material.


Final step:



When we have both the ptsG sequence and the sequence of the red color chromo protein, the next step is to put them inside the plasmid backbone by using restriction enzyme and the ligation enzyme.



1. Restriction enzyme cutting PQE60 : Cut the PQE60 plasmid, ptsG sequence and the red color chromo protein sequence by using the restriction enzymes.



2. Ligase red and ptsG sequences in PQE60: Ligase both the red chromo protein sequence and the ptsG sequence by using the ligation enzyme.



3. Transform recombinant plasmid into dh5α: Transforming our recombinant plasmid into the dh5α is one of the method to confirm whether or not we are successful in the ligation process. If there is any colonies when we are culturing the dh5α, then we can transform our recombinant plasmid into E. coli BL21. The purpose of choosing dh5α is because the dh5α does not contain any plasmids. Hence, enabling us to confirm whether or not we performed the correctly ones in the ligation phase.

4. Transform recombinant plasmid into E. coli BL21: The reason we choose the E. coli BL21 because it can tolerate in high glucose concentration.

After we have done all of the step above, we are able to produce the Chromo Diabetector. Although the steps look easy, but it requires multiple test and tries to get the correct sequence of both the ptsG and the red color chromo protein.
Contact us
 tcutaiwan@gmail.com
No.701, Sec. 3, Zhongyang Rd. Hualien 97004, Taiwan