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

Revision as of 13:39, 13 October 2016

Protein Engineering

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.

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

Red Color Chromoprotein:

1. Making a competent cell:Make E.coli has an ability to take up extracellular genetic material (Red Color Chromoprotein). We need to make the membrane of E.coli bepermeable for our red color chromoprotein.

2. Transformation:after it has become competent cell, now we can transform 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 it 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 the colony which grow in our agar plate and put in into the modified LB media , and grow it inside the incubator for overnight.

5. Measuring the concentration: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.

6. PCR :using the designed primer, we can take the red color chromoprotein sequence from our plasmid

7.purification:after PCR we should purify our red color chromoprotein from the PCR material to get the purify sequence of the red color chromo protein.

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

9.Gel purification: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 BL21, 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 two of them by using ligation enzyme.

3. transform recombinant plasmid into dh5alpha: Transforming our recombinant plasmid into the dh5alpha is one of the method to confirm whether we success in the ligation or not, by growing a colony it. If there is any colony when we culturing dh5alpha means we can transform our recombinant plasmid into E.coli BL21. The purpose of choosing dh5alpha as the bacteria to confirm whether we success in the ligation or not is because dh5alpha does not contain any plasmid, is not like E.coli BL21. Inside the BL21 there is another plasmid beside the plasmid that we want to transform inside it. So, by confirming whether we ligase correctly or not, we using dh5alpha to confirm it.

4. transform recombinant plasmid into 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.

Contact us
tcutaiwan@gmail.com
No.701, Sec. 3, Zhongyang Rd. Hualien 97004, Taiwan

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