Translational Control of Gene Expression Krystal Joan Annand - - PowerPoint PPT Presentation

Engineering a Toggle Switch in Yeast Using Translational Control of Gene Expression

Krystal Joan Annand Brychan Cromwell Lisa Dryburgh Joseph Hoare Justyna Kucia Stephen Lam Christina McLeman Ben Porter Margaret-Ann Seger Liz Threlkeld

Aims

• Design and construct a toggle switch.
• Regulation of switch at translational level is

potentially faster.

• Use yeast as a model organism to increase

the parts available in the Registry of Parts.

• Use mathematical modelling to optimise

the system.

AYESWITCH – How It Works

N-peptide - GFP MS2 - CFP Transcription Translation mRNA DNA Protein MS2 stem loops Bbox galactose Cu2+

Characterisation of the Promoters

The CUP1 Promoter

 The CUP1 promoter shows a clear

dose response indicating that it can be regulated.

 The CUP1 promoter is rapidly

fully induced.

Dose Response of CUP1 Fluorescence units Copper Concentration (μM) Fluorescence units Time / minutes Timed Induction of CUP1

Characterisation of the Promoters

The GAL1 Promoter  The GAL1 showed a high

sensitivity to the inducing agent.

 Expression induction is linear

• ver time and the response is

immediate.

Dose Response of GAL1 Fluorescence units Galactose Concentration (w/v) Fluorescence units Time / minutes Timed Induction of GAL1

Further Characterisation of GAL1

 This confirms that GFP is a very stable protein and

the decay is mainly caused by cell division. Decay of GFP

Fluorescence units Time / minutes

• the sequence of CFP;
• the ‘Regulatory elements’ in CUP1p-[MS2-CFP] using cassette

replacement experiments.

Characterising CUP1p-[MS2-CFP]

 CUP1p-[MS2-CFP] did not function as expected.  To determine the reason we checked:

MS2 - CFP

Checking the Sequence of CFP

 Confirmation that sequence of CFP is correct by cassette

replacement experiment. Yeast cells Yeast cells under fluorescent conditions

Checking the Regulatory Elements

• Replacing the CUP1 promoter with a different version that is

known to work.

• Replacing the fusion protein with a different set of proteins.

Results

 CFP could not be detected in either experiment.  The Bbox sequence or the translational fusion of

MS2 coat protein appears to block protein expression.

Bbox

Characterising Translational Repression

• The translational repression was characterised by trans

expression of the MS2 protein.

1000 2000 3000 4000 5000 100 200 300 400 500 600 700 GFP Fluorescence Methionine (micro molar) GAL1 promoter MET17 promoter N-peptide GFP MS2-protein C-Ub-URA3

High Met = high GFP Low Met = low GFP

How the Switch was Modeled

MET17 promoter GAL1 promoter

DNA mRNA Proteins N-peptide - GFP MS2 - CFP u

Importance of Hill Coefficients

Determines the type of switch - analogue/digital

Promoter Activity Repressor Concentration

n →∞ n = 1 n = 2 n = 4

Solving the Equations for the Steady State

GFP CFP Stable Fixed Point (CFP dominates) Stable Fixed Point (GFP dominates) Unstable Fixed Point

Bifurcation Diagram

Bifurcation Point Stable

C = a dimensionless function of our parameters

GFP

Our Parameters

Parameter Value

(converted to number of molecules, N)

λ1 1.34 x 10-1 s-1 λ2 4.16 x 10-2 s-1 λ3 5.84 x 10-2 s-1 λ4 4.18 x 10-2 s-1 μ1 9.26 x 10-4 s-1 μ2 2.5 x 10-4 s-1 μ3 2.45 x 10-4 s-1 μ4 2.5 x 10-4 s-1 Parameter Value

(converted to number of molecules, N)

K1 5 x 103 K2 1 x 104 K3 5 x 103 K4 1 x 105 T 8.85 x 10-5 s-1 n1 2 n2 1 - 3 n3 4 n4 1 Parameter Value

(converted to number of molecules, N)

K1 5 x 103 K2 1 x 104 K3 5 x 103 K4 1 x 105 T 8.85 x 10-5 s-1 n1 2 n2 1 - 3 n3 4 n4 1

Calculating n2 More Precisely

• Based on a graph of MS2 binding curves in a paper

by Witherell et al (1990) we could calculate the value for the Hill coefficient of the CFP/MS2 stem loop association (n2).

• We reproduced the graph in MATLAB and using

MATLABs curve fitting tool we fit the Hill function for activators to the curve.

• The R2 value was 0.998, suggesting a very good fit

to the curve.

• The result gave n2 = 2.6 ± 0.3.

MS2 concentration (Molar) Fraction of MS2 bound to stem loops MS2 concentration (Molar) Fraction of MS2 bound to stem loops

Do We Get a Switch?

We can get a switch but only under certain circumstances. For example, from a state of high galactose and high methionine in which GFP dominates, we can switch to a CFP dominated state if we actively remove the galactose and methionine from the system.

High Galactose + Methionine = GFP wins No Galactose + No Methionine = CFP wins No Galactose + High Methionine = no proteins High Galactose + No Methionine = competition between proteins!

Parameter Value Range λ1 1.34 x 10-1 1.34 x 10-1±2 λ2 4.16 x 10-2 4.16 x 10-2±2 λ3 5.84 x 10-2 5.84 x 10-2±2 λ4 4.18 x 10-2 4.18 x 10-2±2 μ1 9.26 x 10-4 9.26 x 10-4±2 μ2 2.5 x 10-4 2.5 x 10-4±2 μ3 2.45 x 10-4 2.45 x 10-4±2 μ4 2.5 x 10-4 2.5 x 10-4±2 K1 5 x 103 5.0 x 103±2 K2 1 x 104 1.0 x 104±2 K3 5 x 103 5.0 x 103±2 K4 1 x 105 1.0 x 105±2

Robustness of the Switch

Robustness of the Switch

Parameter Value Range Change λ1 1.34 x 102 1.34 x 102±2 increase by 103 λ2 4.16 x 102 4.16 x 102±2 increase by 104 λ3 5.84 x 102 5.84 x 102±2 increase by 104 λ4 4.18 x 102 4.18 x 102±2 increase by 104 μ1 9.26 x 10-7 9.26 x 10-7±2 decrease by 103 μ2 2.5 x 10-7 2.5 x 10-7±2 decrease by 103 μ3 2.45 x 10-7 2.45 x 10-7±2 decrease by 103 μ4 2.5 x 10-7 2.5 x 10-7±2 decrease by 103 K1 5.0 x 104 5.0 x 104±2 increase by 101 K2 1.0 x 104 1.0 x 104±2 K3 5.0 x 105 5.0 x 105±2 increase by 102 K4 1.0 x 104 1.0 x 104±2 decrease by 101

Robustness of the Switch

Robustness of the Switch

Improving the System - Directed Evolution

A reminder of our parameters

Parameter Original Value Optimal Value Obtained λ1 1.34 x 10-1 1.34 x 102 λ2 4.16 x 10-2 4.16 x 102 λ3 5.84 x 10-2 5.84 x 102 λ4 4.18 x 10-2 4.18 x 102 μ1 9.26 x 10-4 9.26 x 10-4 μ2 2.5 x 10-4 2.5 x 10-4 μ3 2.45 x 10-4 2.45 x 10-4 μ4 2.5 x 10-4 2.5 x 10-4 K1 5 x 103 5 x 103 K2 1 x 104 1 x 104 K3 5 x 103 5 x 103 K4 1 x 105 1x 104

Improving the System - Directed Evolution

Conclusions

 Designed, constructed and tested a novel genetic toggle switch regulated

at the translational level.

 Expressed a novel translational fusion protein.  Demonstrated that translational regulation by coat binding proteins to

mRNA stem loops is a viable design.

 Submitted, tested and characterised four biobricks to the Registry of Parts.

We also tested the biobrick E2050 mOrange .

 Found the optimal parameters for switching behaviour which increased our

success to 98%.

 By modelling directed evolution we successfully found the optimal

parameters for a clearly defined switch.

 Our overall analysis suggests that a translationally regulated genetic toggle

switch is a viable gene circuit/biological machine.

Thank you for listening