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

 
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<td align=center><font size=25>Experiments</font></td>
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<td align=center><font size=25>Experiments<br><br></font></td>
 
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<td align=center><font size=25>Protien Maked</font></td>
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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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<td><span style="font-family:Calibri;text-align:justify;"><font size="5">
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Here’s the experiments that we did to create Chromo Diabetector:<br><br>
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To create a master piece of CHROMO DIABETECTOR, we need to perform some experiment which is cost energy and the patience. In this experiment base on what we have said before at the Design part, we should get the sequence of Red color chromo-protein and PTSG. After we got the sequence for both Red color chromo-protein and PTSG, we need to perform restriction and ligation to the plasmid then transform it to the E.coli.<br><br>
+
Red color chromo protein:<br><br>
−
Here’s the experiment that we did to create Chromo Diabetector:<br>
+
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>
−
Red color chromoprotein:<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>
−
&nbsp;&nbsp;&nbsp;&nbsp;1. Making a competent cell:
+
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>
−
Make E.coli has an ability to take up extracellular genetic material (Red Color Chromoprotein). We need to make the membrane of E.coli be permeable for our red color chromoprotein.  
+
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>
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><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>
−
2. Transformation  
+
10. Get the sequence: Save the red color chromo protein sequence in the -20 fridge for the long term usage.
<br><br>
−
after it has become competent cell, now we can transform Red Color chromo protein which already exists in the PSB1C3 plasmid into the competent cell
+
<I>ptsG</I>:

<br><br>
−
3. Recombinant plasmid culturing
+
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>
−
Now we can culture it in the LB agar media by adding the selective marker and the plasmid that we have already transform into the E.coli.
+
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>
−
4. Plasmid extraction from E.coli  
+
3. PCR: By designing the primer for it, we can get the <I>ptsG</I> sequence.

<br><br>
−
Pick the colony which grow in our agar plate and put in into the modified LB media , and grow it inside the incubator for overnight.
+
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>
−
5. Measuring the concentration  
+


6. Gel purification: Perform the Gel purification to isolate the <I>ptsG</I> sequence from the Gel electrophoresis material.
<br><br>
−
take the plasmid from the incubator and then measure OD value of it, after it reaches a certain OD value we can continue it to the PCR.
+
Final step:

<br><br>
−
6. PCR  
+
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>
−
using the designed primer, we can take the red color chromoprotein sequence from our plasmid
+
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>
−
7.purification
+
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>
−
after PCR we should purify our red color chromoprotein from the PCR material to get the purify sequence of the red color chromo protein
+
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>
−
8.Gel electrophoresis  
+
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>
−
Run the Gel electrophoresis to check whether we get the right sequence of the red color chromoprotein
+
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.
−
9.Gel purification  
+
</font></span></th>
−
Perform the Gel purification of the gel electrophoresis, to get the purify Red color chromoprotein from the gel material
+
−
10. Get the sequence
+
−
Save the red color chromo protein sequence in the -20 fridge for long term usage.
+
−
 
+
−
PTSG
+
−
 
+
−
1. DNA isolation from E.coli  
+
−
Because the sequence of PTSG already exist on the E.coli, what we need to do is just to get the sequence of the PTSG from the E.coli. First, we need to extract the DNA from the E.coli
+
−
2. measuring the concentration  
+
−
After we extract the DNA from the E.coli, we should measure the concentration of the DNA to perform PCR.
+
−
 
+
−
3.PCR  
+
−
After that we run the PCR to get the PTSG sequence, by designing the primer for PCR we can get the sequence.
+
−
4.PCR purification
+
−
perform the PCR purification to clean the PTSG sequence from the PCR material.
+
−
5. Gel electrophoresis  
+
−
then load Our PTSG sequence in the agarose gel to perform the gel electrophoresis, so that we can check wheter we got the right sequence of the PTSG or wrong sequence.
+
−
6. Gel purification
+
−
perform the Gel purification to clean the PTSG sequence from the Gel electrophoresis material.
+
−
Final step
+
−
When we have PTSG sequence and the sequence of the red color chromo protein, next step is to put them inside the plasmid backbone by using restriction enzyme and the ligation enzyme.
+
−
 
+
−
1.restriction enzyme cutting PQE60
+
−
first of all cut the PQE60 plasmid, PTSG sequence and the red color chromo protein sequence using restriction enzyme.
+
−
2.ligase red and ptsg in pqe60
+
−
Ligase three of them by using ligation enzyme
+
−
3. transform recombinant plasmid into E.coli
+
−
Final step is transforming the plasmid inside the E.coli BL21. We choose E.coli BL21 because it can tolerate high glucose concentration.  
+
−
 
+
−
After we have done all of the step, we already create a master piece of Chromo Diabetector. All though it looks easy for the step, but in the reality it’s require many times to get the sequence of both PTSG 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