Team:Lethbridge Canada/Results

From 2012hs.igem.org

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'''RFP with Cells Media at Different Induction Levels'''
'''RFP with Cells Media at Different Induction Levels'''
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Preliminary results
Cultures were grown containing pLacI promoter attached to a Twin Arginine tag signal sequence and fused to red fluorescent protein.  Samples were taken at various time points 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 the supernatant after spinning the cells to a pellet.  We used a constutively expressed construct expressing Red fluorescent protein( J23100 in J61002)
Cultures were grown containing pLacI promoter attached to a Twin Arginine tag signal sequence and fused to red fluorescent protein.  Samples were taken at various time points 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 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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[[Image:Time A.JPG|left|px]][[Image:Time B.JPG|right|px]]
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[[Image:Time A.JPG|left|px]]
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Fluorescents was measured one hour after induction, no difference could be seen between the test and control constructs in the supernatant .
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[[Image:Time B.JPG|right|px]]
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Fluorescents was measured two hours after induction, no difference could be seen between the test and control constructs in the supernatant .
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[[Image:Time C.JPG|left|px]][[Image:Time D.JPG|right|px]]
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[[Image:Time C.JPG|left|px]]
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Fluorescents was measured three hours after induction, no difference could be seen between the test and control constructs in the supernatant .
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[[Image:Time D.JPG|right|px]]
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Fluorescents was measured four hours after induction, no difference could be seen between the test and control constructs in the supernatant .
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Revision as of 06:27, 17 June 2012

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Results

Parts created

We created two new parts, BBa_K736000 (which regulated the transcription of downstream genes) and BBa_K736001 (which was a TAT signal sequence fused to a red fluorescent protein, E1010). We also assembled two parts, BBa_K736002 (which tests the ability of glucose to regulate transcription and uses enhanced RFP as the reporter) and 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 (BBa_K736004) and one part that coded for human insulin chain B (BBa_K736005). We are also submitting the DNA for K331009 which was in its planning stage in the registry.



NameTypeDescriptionLength
BBa_K736000RegulatoryConstitutive Promoter (J23100) with MLC (glucose-regulated) binding site36 bp
BBa_K736001ReporterTAT signal sequence fused to red fluorescent protein (E1010)720 bp
BBa_K736002CompositeConstitutive Promoter with MLC-binding-site-regulated enhanced RFP913 bp
BBa_K736003CompositepLAC-regulated TAT-RFP construct948 bp
BBa_K736004Coding Coding sequence for insulin chain A72 bp
BBa_K736005Coding Coding sequence for insulin chain B99 bp


Proof of Principle

TAT signal sequence

RFP with Cells Media at Different Induction Levels Preliminary results

Cultures were grown containing pLacI promoter attached to a Twin Arginine tag signal sequence and fused to red fluorescent protein. Samples were taken at various time points 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 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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Fluorescents was measured one hour after induction, no difference could be seen between the test and control constructs in the supernatant .

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


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

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























































Glucose Variable Concentrations


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