Difference between revisions of "Part:BBa J45995:Experience"

(→‎Characterization)
(→‎Characterization)
Line 39: Line 39:
  
 
<br style="clear:both;"/>
 
<br style="clear:both;"/>
 +
 +
====William and Mary iGEM 2022====
 +
To test the effectiveness of our improved parts, our team grew the original MIT 2006 construct, our improved sfGFP construct, and our improved RFP construct in <i>E. coli</i> NEB5α in a plate reader. They were grown at 37°C using continuous shaking. For green fluorescence, we used an excitation value of 485 and an emission value of 528.  For red fluorescence, we used an excitation value of 584 and an emission value of 610. The values for green fluorescence are reported below. For information about red fluorescence, see parts page BBa_K4174001.
 +
 +
https://static.igem.wiki/teams/4174/wiki/normalized-green-fluorescence.png
 +
 +
As seen in the graph above, both the sfGFP and MIT GFP constructs enter stationary phase right before 14 hours, but our improved sfGFP circuit is much more fluorescent. The other constructs are our RFP construct and untransformed <i>E. coli</i> cells, both of which serve as negative controls for green fluorescence.
 +
 +
 +
https://static.igem.wiki/teams/4174/wiki/improveapart-smaller.png
 +
 +
As seen in the image above, qualitative results reveal that our improved constructs are more fluorescent than the original construct. Here, sfGFP is on the far right, and is visibly more brightly green that the original GFP construct.

Revision as of 18:50, 8 October 2022

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_J45995

Stationary phase dependent fluorescence.

User Reviews

UNIQd23a7c3fae6e2ea6-partinfo-00000000-QINU

•••••

Reshma Shetty

BBa_J45995 produced fluorescence only in stationary phase.

UNIQd23a7c3fae6e2ea6-partinfo-00000003-QINU

Characterization

Transcriptional control of GFP generator

[Note: BBa_J45995 is a composite part of BBa_J45992 and BBa_E0840.]

Growth phase dependent transcriptional control devices
We successfully designed, constructed and tested transcriptional control devices for constitutive, stationary phase dependent and exponential phase dependent protein production (A-C). To test and verify function of our three transcriptional control devices, we assembled each control device with the GFP protein generator BBa_E0840 and monitored the fluorescence of E. coli cultures with each device over time. For each device, we plot the change in fluorescence per unit time (normalized GFP synthesis rate) versus the cell density (OD600nm) (D). The constitutive transcriptional control device produced a high GFP synthesis rate irrespective of cell density. The stationary phase transcriptional control device produced a low initial GFP synthesis rate which increased with culture cell density. The exponential phase transcriptional control device produced an initially high GFP synthesis rate which dropped off as cell density increased. Data shown are averages of triplicate measurements of cultures grown from three individual colonies of each device. Error bars are the standard deviation of the three individual cultures.


William and Mary iGEM 2022

To test the effectiveness of our improved parts, our team grew the original MIT 2006 construct, our improved sfGFP construct, and our improved RFP construct in E. coli NEB5α in a plate reader. They were grown at 37°C using continuous shaking. For green fluorescence, we used an excitation value of 485 and an emission value of 528. For red fluorescence, we used an excitation value of 584 and an emission value of 610. The values for green fluorescence are reported below. For information about red fluorescence, see parts page BBa_K4174001.

normalized-green-fluorescence.png

As seen in the graph above, both the sfGFP and MIT GFP constructs enter stationary phase right before 14 hours, but our improved sfGFP circuit is much more fluorescent. The other constructs are our RFP construct and untransformed E. coli cells, both of which serve as negative controls for green fluorescence.


improveapart-smaller.png

As seen in the image above, qualitative results reveal that our improved constructs are more fluorescent than the original construct. Here, sfGFP is on the far right, and is visibly more brightly green that the original GFP construct.