Difference between revisions of "Part:BBa K1980008"

Latest revision as of 21:12, 24 October 2016

pCopA CueR sfGFP/ feedback pCopA sfGFP

Description

This construct contains pCopA (a copper sensitive promoter) with CueR (a transcription factor that binds onto pCopA) and a sfGFP (a variant of the GFP) downstream. The idea behind this design is that expression of CueR under the control of the promoter it regulates would produce a positive feedback loop, making the construct more sensitive to different copper concentrations.

CueR can act as both a positive and negative regulator and our results seem to indicate that negative regulation may be occurring here.

Sequence and Features:

Usage and Biology

Our project was to investigate a probiotic treatment for the copper-accumulation disorder: Wilson's disease. This required a system able to detect dietary copper, ideally in the range over which copper concentration changes after a meal (around 5-10μM).

E. coli cells use a protein called CueR to regulate the cytoplasmic copper concentration. CueR is a MerR-type regulator with an interesting mechanism of action whereby it can behave as a net activator or a net repressor under different copper concentrations through interaction with RNA polymerase⁽¹⁾. CueR forms dimers consisting of three functional domains (a DNA-binding, a dimerisation and a metal-binding domain). The DNA binding domains bind to DNA inverted repeats called CueR boxes with the sequence:

CCTTCCNNNNNNGGAAGG

This box is present at the promoter regions of the copper exporting ATPase CopA, some molybdenum cofactor synthesis genes and the periplasmic copper oxidase protein CueO.⁽²⁾

We designed this part with CueR produced from the pCopA promoter it controls so it should form a feedback loop. The following picture is a representation of how the construct works, theoretically in vivo:

Schematic of how BBa K1980008 was intended to operate. The feedback may not be positive here

Experience

We cloned pCopA CueR sfGFP/ feedback pCopA sfGFP from a gBlock into the shipping plasmid pSB1C3. E. coli strain MG1655 was transformed using the specific recombinant plasmid and a 5ml culture of a transformed colony was grown overnight. The functionality of the construct was tested using plate reading, flow cytometry and microscopy

Plate Reader

The results of a plate reader experiment with different copper concentrations after four hours. Error bars show standard deviation of four repeats

We tested the promoter with the fluorescent protein sfGFP (a form of GFP with excitation/emission maxima at 485-512nm and 520nm).

To account for the number of cells present at different copper concentrations and different times we measured the optical density (OD) as a proportional measure of the number of cells present.

A range of copper concentrations were prepared from stock solutions. A large volume plate was then prepared with 10μl of copper solution, 10μl of overnight culture and 980μl of broth with antibiotic. This resulted in a 1 in 100 dilution of the copper solutions prepared.

This large-volume plate was then centrifuged to mix the solutions and then 200μl transferred to a small-volume plate with a clear lid and then placed in the plate reader.

Four biological repeats of this part at ten different copper concentrations were measured as well as four repeats of a negative control (The NC part from the interlab study) and a positive control (The constitutive GFP, PC part from the Interlab study).

The plate reader measured the fluorescence and OD every ten minutes for at least 12 hours, shaking between measurements.

Flow cytometry

Flow cytometry of this part at different copper concentrations

Flow cytometry is a technique whereby cells are passed individually into the path of a beam of light. The frequency range of incident light can be adjusted to allow excitation of specific fluorophores in the sample of cells. Downstream detectors can measure fluorescent emission, and this data can be used to quantify the amount of fluorophore in each cell.

To ensure comparability of experiments, all cells were grown for 3-4hrs (until entrance into the exponential growth phase) at 37°C and 225rpm shaking in the presence of the inducer before measurement. This allowed adequate time for activation of expression by the promotor systems. The negative control used in all cases were MG1655 bacteria containing an empty shipping plasmid. As these did not contain any fluorescent molecules, this population could be used to set the negative “gate” (i.e. the background fluorescence of the bacterial cells). Although the experiments were tedious as every sample had to be measured manually, the results were of remarkably high quality, were clearly interpretable, and fit very well with the other experimental data.

Microscopy

Microscopy performed on this part at different copper concentrations. Fluorescent, differential interference contrast channels and a composite shown

Microscopy was done in order to visually confirm the plate reader and flow cytometer experiments.

The experiment started with 5ml overnight cultures containing the appropriate antibiotic. Then in the morning 100μl of each colony was pipetted into 5ml of fresh LB with antibiotic and a range of copper concentrations and grown till the OD reached 0.4-0.6.

A flask of 1% agarose made with MilliQ was melted. 200μl of this was placed on a slide between two coverslips, flattened to get a nice smooth surface where the bacteria are immobile. 20μl of the culture are then added. The slide was then be viewed under a fluorescence microscope.

After finding the correct focal plane the slide was moved to find as many cells as possible to image. After focusing again an image of the DIC and fluorescence channels was obtained.

Conclusions

We designed this part in order to study how CueR operates under a feedback loop. If CueR is acting as a net activator, this should in theory act as a positive feedback system whereby a small amount of copper stimulation produces more CueR causing greater activation. Should CueR be acting as a net repressor this system would be less sensitive than the system with CueR constitutively expressed divergent from pCopA (BBa_K1980006) and this appears to be what we discovered.

References

(1) Danya J. Martell, Chandra P. Joshi, Ahmed Gaballa, Ace George Santiago, Tai-Yen Chen, Won Jung, John D. Helmann, and Peng Chen (2015) “Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription” Proc Natl Acad Sci U S A. 2015 Nov 3; 112(44): 13467–13472.

(2) Yamamoto K, Ishihama A. (2005) “Transcriptional response of Escherichia coli to external copper.” Mol Microbiol. 2005 Apr;56(1):215-27.

Author: Sam Garforth and Andreas Hadjicharalambous