Composite
Phosphoryl

Part:BBa_K3773515:Experience

Designed by: Avery Bradley   Group: iGEM21_William_and_Mary   (2021-10-14)

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.

iGEM21_William_and_Mary

This part was successfully sequenced by Epoch Life Science and sequence-confirmed using Benchling tools.

Our initial functional confirmation of this circuit used the same process as for BBa_K3773514; it was successful.


We transformed this circuit into cells alone or alongside pBbB8k-csg-amylase, whose effect on this circuit's expression we hoped to quantify through a change in fluorescence.

T--William_and_Mary--FigLegendRegistry.png T--William_and_Mary--Results_WM21_015_rawfluor.png T--William_and_Mary--Results_WM21_015_nummolecs.png

The following cultures were grown up: one flask of untransformed competent E.coli NEB 5-alpha cells (Untransformed), one flask of phosphorylation sensor WM21_015 alone (Sensor Circuit), and two flasks of WM21_015 co-transformed with pBbB8k-csg-amylase (arabinose-inducible curli fiber circuit) (Sensor + Test). The sensor circuit and co-transformations were also in E.coli NEB 5-alpha cells. T = -1 represents measurements taken from these cultures after a growth period of approximately 12 hours, before making subcultures. T = 0 represents measurements taken directly after making subcultures. One flask of WM21_015 co-transformed with pBbB8k-csg-amylase was then induced (Sensor + Test - Induced), while the other remained uninduced (Sensor + Test - Uninduced). T = 1 represents measurements taken 1 hour after the induction step. Measurements were also taken for T=6, T=12, T=24, and T=48 hours post-induction. This process was repeated a total of three times, and the individual recordings are displayed as circles (n=3). The average measurements for each experimental group are displayed as stars and are connected by a line. “Number of molecules” refers to the number of sfGFP molecules per cell, calculated from fluorescence and OD values. P-values for comparison are available on the Results page.

Results:

WM21_015 (our phosphorylation sensor) is based on the EnvZ/OmpR system. This system responds to changes in osmolarity of the environment by phosphorylation of the OmpR protein which then triggers production of either OmpC in high molarity states or OmpF in low osmolarity states. This circuit utilizes the ompC promoter to drive production of sfGFP. Therefore, this fluorescence output is indicative of phosphorylation rates. 

Our team expected that our phosphorylation sensor would decrease in fluorescence output when an additional circuit was added to the host due to the additional burden caused by the results. The results we received were not exactly as predicted. This circuit actually produced more fluorescence when induced than when uninduced; however, there were no statistically significant differences between these values over time. 

Due to the increase in fluorescence in the induced vs the uninduced, the osmolarity of the system must have increased. Given that the circuit our team used as our test plasmid was a biofilm producing plasmid, our team hypothesizes that biofilm production created an increase in the osmolarity of the surrounding environment. This increase in osmolarity triggered the EnvZ/OmpR system to increase phosphorylation rates. Overall, there was no significant difference in the amount of fluorescence output between our sensor circuit with an additional plasmid, induced or uninduced. 

In summary:

  • Our team received unexpected results for our phosphorylation sensor
  • There was no statistically significant difference between expression between the induced and uninduced sensors
  • This circuit produced more fluorescence when induced than when uninduced


User Reviews

UNIQ4f2d413cc629862e-partinfo-00000000-QINU UNIQ4f2d413cc629862e-partinfo-00000001-QINU