Team:Lethbridge Canada/Results

From 2012hs.igem.org

(Difference between revisions)
(→Proof of Principle)
(→Proof of Principle)
Line 58: Line 58:
Preliminary results
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 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 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)
<br>
<br>
Line 94: Line 94:
-
Fluorescents was measured two hours after induction, no difference could be seen between the test and control constructs in the supernatant .  
+
Fluorescence was measured two hours after induction, no difference could be seen between the test and control constructs in the supernatant .  
Line 117: Line 117:
-
Fluorescents was measured three hours after induction, no difference could be seen between the test and control constructs in the supernatant .  
+
Fluorescence was measured three hours after induction, no difference could be seen between the test and control constructs in the supernatant .  
Line 139: Line 139:
-
Fluorescents was measured four hours after induction, no difference could be seen between the test and control constructs in the supernatant .  
+
Fluorescence was measured four hours after induction, no difference could be seen between the test and control constructs in the supernatant .  
<br>
<br>
Line 184: Line 184:
-
Fluorescents was measured three hours after innoculation, no emissions were observed.   
+
Fluorescence was measured three hours after innoculation, no emissions were observed.   
<br>
<br>
Line 205: Line 205:
-
Fluorescents was measured four and a half hours after innoculation, no emissions were observed.   
+
Fluorescence was measured four and a half hours after innoculation, no emissions were observed.   
<br>
<br>
Line 225: Line 225:
-
Fluorescents was measured six hours after innoculation, no emissions were observed.   
+
Fluorescence was measured six hours after innoculation, no emissions were observed.   
<br>
<br>

Revision as of 06:52, 17 June 2012

HP.JPG
Home The Team The Project Results Human Practices Notebook Safety



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


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 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)


px






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







px





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








px





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






px






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













Glucose Variable 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.



px





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




px







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





px





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



Retrieved from "http://2012hs.igem.org/Team:Lethbridge_Canada/Results"