Team:Lethbridge Canada/Results

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=='''Insulin Red Fluorescent Protein Proof of Principle'''==
=='''Insulin Red Fluorescent Protein Proof of Principle'''==

Revision as of 06:56, 17 June 2012

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Contents

Results

Parts created

We created two new parts, [http://partsregistry.org/Part:BBa_K736000 BBa_K736000] (which regulated the transcription of downstream genes) and [http://partsregistry.org/Part:BBa_K736001 BBa_K736001] (which was a TAT signal sequence fused to a red fluorescent protein, E1010). We also assembled two parts, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K736002 BBa_K736002] (which tests the ability of glucose to regulate transcription and uses enhanced RFP as the reporter) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K736003 BBa_K736003] (which tests for the ability of the TAT sequence to cause secretion of protein outside of the cell and also uses RFP as a reporter). We also created one part that coded for human insulin chain A ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K736004 BBa_K736004]) and one part that coded for human insulin chain B ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K736005 BBa_K736005]). We are also submitting the DNA for [http://partsregistry.org/Part:BBa_K331009 K331009] which was in its planning stage in the registry.



NameTypeDescriptionLength
[http://partsregistry.org/Part:BBa_K736000 BBa_K736000]RegulatoryConstitutive Promoter (J23100) with MLC (glucose-regulated) binding site36 bp
[http://partsregistry.org/Part:BBa_K736001 BBa_K736001]ReporterTAT signal sequence fused to red fluorescent protein (E1010)720 bp
[http://partsregistry.org/Part:BBa_K736002 BBa_K736002]CompositeConstitutive Promoter with MLC-binding-site-regulated enhanced RFP913 bp
[http://partsregistry.org/Part:BBa_K736003 BBa_K736003]CompositepLAC-regulated TAT-RFP construct948 bp
[http://partsregistry.org/Part:BBa_K736004 BBa_K736004]Coding Coding sequence for insulin chain A72 bp
[http://partsregistry.org/Part:BBa_K736005 BBa_K736005]Coding Coding sequence for insulin chain B99 bp



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Insulin Red Fluorescent Protein Proof of Principle

TAT signal sequence

RFP with Cells Media at Different Induction Levels

Preliminary results

Cultures were grown containing LacI promoter attached to a twin arginine tag (TAT) signal sequence and fused to a red fluorescent protein (E1010). Samples were taken at various time points and examined for fluorescence when excited at 586nm with an expected emission at 607nm. We used a fluorescence spectrophotometer to measure the red fluorescent protein present in the supernatant after spinning the cells to a pellet. We used a constutively expressed construct expressing Red fluorescent protein( J23100 in J61002)


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Fluorescence was measured one hour after induction, no difference could be seen between the test and control constructs in the supernatant .







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Fluorescence was measured two hours after induction, no difference could be seen between the test and control constructs in the supernatant .








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Fluorescence was measured three hours after induction, no difference could be seen between the test and control constructs in the supernatant .






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Fluorescence was measured four hours after induction, no difference could be seen between the test and control constructs in the supernatant .

Glucose Detection Concentrations

Preliminary Results

Cultures were grown containing constitutively expressed promoter J23100 attached to an MlC binding sight sequence and a red fluorescent protein. Samples were inoculated in varying concentrations of Glucose (0=0 mg/dl, 20= 12.5mg/dl, 40=25mg/dl, 80=50mg/dl, 160=100mg/dl, 320=200mg/dl, 1600=1000mg/dl). Samples were taken after 1.5 hour time points post inoculation and examined for fluorescents when excited at 586nm with an expected emission at 607nm. We used a fluorescents spectrophotometer to measure the red fluorescent protein present in cell lysate after spinning the cells to a pellet and re-suspending in 8M urea.



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Fluorescence was measured three hours after innoculation, no emissions were observed.




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Fluorescence was measured four and a half hours after innoculation, no emissions were observed.





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Fluorescence was measured six hours after innoculation, no emissions were observed.