Difference between revisions of "Part:BBa K1182000:Experience"

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__NOTOC__
 
__NOTOC__
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
how you used this part and how it worked out.
 
how you used this part and how it worked out.
  
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'''===Applications of BBa_K1182000==='''
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===Applications of BBa_K1182425===
  
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To test the function of our split reporter, we collect the data of enzyme activity over time. We exploit the enzyme’s ability to convert ortho-Nitrophenyl-β-galactoside (ONPG), a colorless substance, into ortho-nitrophenol, a yellow substance. We then use a spectrophotometer to quantisize the absorbance of the solution.
+
=='''Activity Assay of The Split Reporter'''==
  
 +
To test the function of our split reporter, we collect the data of enzyme activity over time. We exploit the enzyme’s ability to convert ortho-Nitrophenyl-β-galactoside (ONPG), a colorless substance, into ortho-nitrophenol, a visible yellow substance. We then use a spectrophotometer to quantisize the absorbance of the solution.
  
  
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== '''Experimental setup & protocol''' ==
 
  
 +
'''Experimental setup & protocol'''
  
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1. The alpha and omega fragment of β-galactosidase was cloned into pQE-80L and pQE-81L, respectively
+
1. The α and ω fragment of β-galactosidase was cloned into pQE-80L and pQE-81L, respectively
  
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2. The alpha and omega fragment was then expressed in TOP10 E.coli
+
2. The α and ω fragment was then expressed (in TOP10 E.coli) and purified using His tagged protein purification method
  
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3. An equal molar of both the alpha and omega peptide, as well as the diluted full length β-galactosidase, then added into an eppendorf tube and incubated at room temperature on an orbital rocker for 1 hour.  
+
3. Add an equal molar of both the α and ω peptide to eppendorf tube #1 - #4; α fragment to eppendorf tube #5 - #8; ω fragment to eppendorf tube #9 - #12; and diluted full length β-galactosidase to tube #13 - #16. Incubate those tubes at room temperature on an orbital rocker for 1 hour.  
  
 
4. At time zero, 20µL of ONPG (4mg/mL) was added into each tube
 
4. At time zero, 20µL of ONPG (4mg/mL) was added into each tube
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'''== Result =='''
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'''Result'''
  
 +
[[File:Activity Assay New.jpg]]
 +
 +
 +
 +
'''Interpretation'''
 +
 +
The full length β-galactosidase reaction mix works as a positive control while both the α-only and ω-only reaction mix works as a negative control.
 +
 +
The split reporter have the activity of full length β-galactosidase enzyme, while none of the α-only nor the ω-only have the enzymatic activity. This data suggests that the peptide complementation needs to occur in order to generate enzymatic activity. Previous study shows that the peptide needs many minutes to form the tetrameric structure which have the enzymatic activity. That’s why we incubate them for 1 hour after mixing the α and ω fragment.
 +
 +
The data also shows that the split reporter needs longer timer to digest the same amount of ONPG compared to full length β-galactosidase. This data suggest that our split reporter works as expected.
  
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[[File:Activity Assay.jpg]]
 
  
 
===User Reviews===
 
===User Reviews===
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<!-- End of the user review template -->
 
<!-- End of the user review template -->
 
<!-- DON'T DELETE --><partinfo>BBa_K1182000 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K1182000 EndReviews</partinfo>
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{|width='80%' style='border:1px solid gray'
 +
|-
 +
|width='10%'|
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<partinfo>BBa_K1182000 AddReview 4</partinfo>
 +
<I>mhanifi/thidayat</I>
 +
|width='60%' valign='top'|
 +
The results indicate that the split reporter system indeed have the enzymatic activity eventhough the reaction rate is slower. It is also proven that the split reporter still possess the enzymatic activity after 28 days storage in 4 degree celcius freezer.
 +
|};
 +
 +
=='''Stability Assay of The Split Reporter'''==
 +
 +
We perform a stability assay to test the enzyme’s activity after freezing and storage in 4ᵒ C freezer for a different length of time.
 +
 +
 +
'''Experimental setup & protocol'''
 +
 +
1. Add α and ω fragment of β-galactosidase to different eppendorf tube.
 +
 +
2. Store those two tubes in 4ᵒC freezer for different length of time (2 days, 7 days, 14 days, 21 days and 28 days)
 +
 +
3. Thaw the tube after stored in different length of time
 +
 +
4. Add an equal molar of both the α and ω peptide to an eppendorf tube. Incubate those tubes at room temperature on an orbital rocker for 1 hour.
 +
 +
5. At time zero, 20µL of ONPG (4mg/mL) was added into each tube
 +
 +
6. The eppendorf tubes then incubated at room temperature for 3 hours
 +
 +
7. The reaction was then terminated by adding 50µL 1M Na2CO3
 +
 +
8. The absorbance is the analysed using 420 nm light
 +
 +
9. Results are expressed as percent signals obtained from freshly expressed enzyme after 3 hours of reaction
 +
 +
 +
 +
'''Result'''
 +
 +
[[File:Stability Assay.jpg]]
 +
 +
 +
 +
'''Interpretation'''
 +
 +
The activity of beta-galactosidase enzyme is still above 95% after 28 days of storage. This data suggest that split reporter can be stored for a quite long time in 4ᵒC.
 +
 +
 +
===User Reviews===
 +
<!-- DON'T DELETE --><partinfo>BBa_K1182425 StartReviews</partinfo>
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<!-- Template for a user review
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{|width='80%' style='border:1px solid gray'
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|-
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|width='10%'|
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<partinfo>BBa_K1182425 AddReview number</partinfo>
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<I>Username</I>
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|width='60%' valign='top'|
 +
The results indicate that the split reporter system indeed have the enzymatic activity eventhough the reaction rate is slower. It is also proven that the split reporter still possess the enzymatic activity after 28 days storage in 4 degree celcius freezer.
 +
|};
 +
<!-- End of the user review template -->
 +
<!-- DON'T DELETE --><partinfo>BBa_K1182425 EndReviews</partinfo>

Latest revision as of 18:12, 27 September 2013

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1182425

Activity Assay of The Split Reporter

To test the function of our split reporter, we collect the data of enzyme activity over time. We exploit the enzyme’s ability to convert ortho-Nitrophenyl-β-galactoside (ONPG), a colorless substance, into ortho-nitrophenol, a visible yellow substance. We then use a spectrophotometer to quantisize the absorbance of the solution.


Experimental setup & protocol

1. The α and ω fragment of β-galactosidase was cloned into pQE-80L and pQE-81L, respectively

2. The α and ω fragment was then expressed (in TOP10 E.coli) and purified using His tagged protein purification method

3. Add an equal molar of both the α and ω peptide to eppendorf tube #1 - #4; α fragment to eppendorf tube #5 - #8; ω fragment to eppendorf tube #9 - #12; and diluted full length β-galactosidase to tube #13 - #16. Incubate those tubes at room temperature on an orbital rocker for 1 hour.

4. At time zero, 20µL of ONPG (4mg/mL) was added into each tube

5. The eppendorf tubes then incubated at room temperature for different length of time (30 min, 90 min, 3 hours, and 19 hours).

6. The reaction was then terminated by adding 50µL 1M Na2CO3

7. The absorbance is the analysed using 420 nm light


Result

Activity Assay New.jpg


Interpretation

The full length β-galactosidase reaction mix works as a positive control while both the α-only and ω-only reaction mix works as a negative control.

The split reporter have the activity of full length β-galactosidase enzyme, while none of the α-only nor the ω-only have the enzymatic activity. This data suggests that the peptide complementation needs to occur in order to generate enzymatic activity. Previous study shows that the peptide needs many minutes to form the tetrameric structure which have the enzymatic activity. That’s why we incubate them for 1 hour after mixing the α and ω fragment.

The data also shows that the split reporter needs longer timer to digest the same amount of ONPG compared to full length β-galactosidase. This data suggest that our split reporter works as expected.


User Reviews

UNIQ54677e1b856901bc-partinfo-00000000-QINU UNIQ54677e1b856901bc-partinfo-00000001-QINU

••••

mhanifi/thidayat

The results indicate that the split reporter system indeed have the enzymatic activity eventhough the reaction rate is slower. It is also proven that the split reporter still possess the enzymatic activity after 28 days storage in 4 degree celcius freezer.

;

Stability Assay of The Split Reporter

We perform a stability assay to test the enzyme’s activity after freezing and storage in 4ᵒ C freezer for a different length of time.


Experimental setup & protocol

1. Add α and ω fragment of β-galactosidase to different eppendorf tube.

2. Store those two tubes in 4ᵒC freezer for different length of time (2 days, 7 days, 14 days, 21 days and 28 days)

3. Thaw the tube after stored in different length of time

4. Add an equal molar of both the α and ω peptide to an eppendorf tube. Incubate those tubes at room temperature on an orbital rocker for 1 hour.

5. At time zero, 20µL of ONPG (4mg/mL) was added into each tube

6. The eppendorf tubes then incubated at room temperature for 3 hours

7. The reaction was then terminated by adding 50µL 1M Na2CO3

8. The absorbance is the analysed using 420 nm light

9. Results are expressed as percent signals obtained from freshly expressed enzyme after 3 hours of reaction


Result

Stability Assay.jpg


Interpretation

The activity of beta-galactosidase enzyme is still above 95% after 28 days of storage. This data suggest that split reporter can be stored for a quite long time in 4ᵒC.


User Reviews

UNIQ54677e1b856901bc-partinfo-00000003-QINU UNIQ54677e1b856901bc-partinfo-00000004-QINU