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

Revision as of 21:42, 8 October 2022

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Applications of BBa_J45995

Stationary phase dependent fluorescence.

User Reviews

UNIQ11a9998493e53d78-partinfo-00000000-QINU

UNIQ11a9998493e53d78-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

BBa_K4174001

To test the effectiveness of our improved parts, our team grew the original construct (BBa_J45995), our improved mRFP1 construct, and our improved sfGFP construct (BBa_K4174001) in E. coli NEB5α in a plate reader. They were grown at 37°C using continuous shaking. For red fluorescence measurements, we used an excitation value of 584 nm and an emission value of 610 nm. For green fluorescence measurements, we used an excitation value of 485 nm and an emission value of 528 nm. The values for red fluorescence are reported below. For information about green fluorescence measurements, see parts page BBa_K4174002.

Based on the graph above, the bacterial cells engineered with our mRFP1 construct appear to have entered stationary phase around 16 hours. As seen in the graph, our mRFP1 construct produces more red fluorescence than the original circuit. The other measurements taken are for our sfGFP construct and untransformed E. coli NEB5α cells, both of which serve as negative controls for red fluorescence.

As seen in the image above, qualitative results reveal that our mRFP1 construct produces more red fluorescence than the original construct. Here, our mRFP1 construct is on the far left, and is visibly more red that the original GFP construct.

BBa_K4174002 To test the effectiveness of our improved parts, our team grew the original MIT 2006 construct, our improved sfGFP construct (BBa_K4174002), and our improved mRFP1 construct (BBa_K4174001) 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.

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.

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.