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From 2014.igem.org

Background

New novel methods must be first tested for viability against other existing methods. Our project is no different. In order, to gauge the effectiveness and accuracy of our method we choose test proteins that are well studied and characterized. Therefore, BINDI and several of its mutant variants that have been well studied were chosen. The first step of our project was to replicate the results of the studies on BINDI and its variants by repeating the stability test experiments presented in "the paper." After verifying the results of "the paper", we proceeded to construct our degron protein constructs and expressed them in yeast cells containing an inducible mechanism for the expression of green fluorescence protein. Subsequently, the fluorescent emission of each cell is measured as higher fluorescent corresponds to higher test protein stability.

Our System

Components of the Degron Construct

Test Protein

The test protein that must be chosen in testing a novel and new system must be a protein that has been well studied and rigorously examined through other existing and well established protein stability testing methods. Therefore,our team decided to use the protein known as BINDI.

Method

The essential process of our system involves cloning and manufacturing of a plasmid in E. coli . Once, the plasmids have been constructed and verified in E. coli they are transformed into S. cerevisiae. The plasmid constructs are then expressed. Following several days of growth the yeast cultures are passed through a Flow Cytometer and the fluorescence of each cell is measured. Higher fluorescence is associated with higher expression of the protein of interest which in-turn is indicative of higher protein stability.

Cloning in Escherichia coli

There are five possible degron constructs corresponding to five different positions the degron can take in our construct. The first step is to insert our protein into each of the five constructs and verify it.

Preparation and Passaging of Saccharomyces cerevisiae

Once, plasmids of the five possible degron constructs have been cloned with our three test proteins, they are subsequently transformed in PyE1 a strain of S. cerevisiae with the ability to produce green fluorescent proteins with the proper promoter protein such as Gal4 which is a part of our degron construct. Following the transformation, the cells are plated onto plates with on a Selective Dropout C-Uracil media and incubated at 30^oC for 2 days. After two days, three colonies are chosen and added to an overnight culture of 3mL Selective Dropout Media C-Uracil and 2% Glucose then incubated for another two days at 30^oC. After two days of incubation, a 20-50uL aliquot of each culture is "passaged" into another 3mL culture prepared in the same manner as before and incubated for the same duration and temperature as the previous culture. The passaging is done several times after each passage after the second passage, a glycerol stock is prepare from the culture and Flow Cytometry is run on the culture.

The purpose of passaging is to gradually remove excess copies of the plasmid constructs. Excess copies, exceeding one per cell will lead to multiple fold increase in the expression of the degron protein construct. As a result of this, GFP expression will also be increased thus reducing the viability and accuracy of the Flow Cytometry measurements conducted on each cell culture.

Relative Stability Analyzed via Flow Cytometry

Mutagenesis through Error Prone Polymerase Chain Reactions

Selecting Stable Variants through Fluorescence Activated Cell Sorting

Results

Protein stability analysis using Circular Dichroism

Our system was verified using Circular Dichroism (CD) analysis. A scan of the protein in solution in PBS was scanned across a variety of wavelengths to find the signal minima that would best indicate the state of folding. An equivalent concentration of protein in concentrated guanidinium chloride (GdmCL), a powerful chaotropic agent, was then prepared to be mixed in to our sample. This solution was then added to the sample in small increments, allowing us to measure the CD signal at increasing concentrations of GdmCl while maintaining a constant concentration of the protein being tested. The concentration of GdmCl at which the CD signal was half of its initial value was recorded. A higher concentration of GdmCl being required to half denature a protein indicates greater stability.

Protein expression analysis using SDS-PAGE

Samples of our protein of interest were taken at various points during the purification cycle. These samples were then run through gel electrophoresis to determine the relative amounts of protein produced by the cells. Larger bands were indicative of greater expression.

Protein stability analysis using Degron Constructs and Flow Cytometry

Mutagenesis Results and Mutant Variant Analysis

Future Plans

Examination of more proteins

Further evolving more stable variants of existing proteins

Submitted Parts

Degron (BBa_K1408000)

The Degron is an unstable protein domain that, when fused to a protein, acts as a source of instability. The presence of the Degron leads to degradation of the protein by the cell via ubiquitination. This is a new part we are submitting to the registry.

Gal4-VP16 Transcriptional Activator (BBa_K1408001)

This part consists of our Gal4-VP16 fusion protein, which is a translational activator. Gal4 protein binds to a Gal1 promoter, while VP16 recruits transcription machinery, promoting transcription to anything under the Gal1 promoter. The two must be co-localized to work as a transcriptional activator.

Gal4-Degron-VP16 (BBa_K1408002)

This part is a combination of our previous two parts submitted to the registry. By utilizing this system, the relative stabilities of proteins can be compared. A sequence for a protein inserted between the Degron and VP16 will produce a fusion protein which acts as a transcriptional activator for anything under a Gal1 promoter. If the protein inserted is unstable it will be degraded by the cell and the Gal4-VP16 transcriptional activator will no longer function. If a quantifiable marker protein such as Green Fluorescent Protein (GFP) is under Gal1 then the level of GFP output of the cell is related to the stability of the inserted protein.