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From 2014.igem.org

Background

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, as the fluorescent emission of each cell is measured as higher fluorescent corresponds to higher test protein stability.

Our System

Components of the Degron Construct

Our experiment utilizes 5 different Degron constructs:
-Deg0: This construct contains only the Gal4-VP16 transcriptional activator complex with the protein of interest in between the two (shortened as Gal4-Protein-VP16).
-Deg1: This construct contains the Degron in front of our Gal4-Protein-VP16 complex.
-Deg2: This construct contains the Degron in between Gal4 and the protein in our Gal4-Protein-VP15 complex.
-Deg3: This construct contains the Degron in between the protein and VP16 in our Gal4-Protein-VP15 complex.
-Deg4: This construct contains the Degron at the end of our Gal4-Protein-VP16 complex.

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 accepted protein stability testing methods. Therefore,our team decided to use the protein known as BINDI. BINDI and two of its less stable variants, BbpD04 and BbpD04.3 were studied and examined in "CITE THE BINDI PAPER." Many of the individuals who were cited in the paper as well as the lab that did the research was done in was a nearby lab at the University of Washington. Therefore, it was convient for us to contact members of the Baker Lab and speak with them about the protein we intended on using as well as accquiring samples of the protein's DNA code.

PYE1 a strain of Saccharomyces cerevisiae

The lynchpin of our project is the usage of flow cytometry and fluorescence activated cell sorting for high throughput protein stability analysis. However, both analytical systems, flow cytometry and F.A.C.S. require fluorescence emmission. Therefore we need a method of generating fluorescence within our cells in a way that also gives us insight into protein stability. Each degron construct contains a Gal4 promoter which can bind to an upstream activating site, Gal1, that induces downstream expression of something. It just so happens that PYE1, this something happens to be Green Fluorescent Proteins. Protein expressions in PYE1, will allow us to generate GFP relative to the amount of degron protein construct that exist within the cell. The more stable the degron protein construct is, the more likely it is that more GFP will be expressed. This relationship between stability and GFP forms the basis from which we will measure the relative protein stability of our degron constructs as well as the protein of interest degron construct.

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. Four of the five vectors for our protein of interest DNA code contains contain a degron (Deg1-4) as well as an EcoRI and Nhel111 restriction enzyme cutsite between Gal4 and VP16. Furthermore, the vector also contains a region that encodes Ampicillin resistance as well as autotrophic region that encodes for uracil synthesis. The DNA that encodes our protein of interest, the insert, is amplified to include both cutsites through a polymerase chain reaction. In a subsequent step the amplified fragment is then digested and then ligated with the appropriate vector and transformed into chemically competent E.coli wither XL-1 Blue or XL10-Gold strains. Similarly, Deg0(no degron) vector has EcoRI along with a Hind111 cutsite. Using the different cutsites our DNA fragment is prepared using PCR and ligated into the Deg0 vector and then transformed. Once the transformation is complete, the cells are plated onto LB-agar plates supplimented with ampicillin in order to ensure that all E.coli colonies contain our recombinant plasmid. After being grown for a day, several colonies are swiped and added to an overnight culture. The overnight culture is grown overnight and their recombinant plasmid is harvested and sequenced (Sanger sequencing is used through Genewiz Inc.). If the plasmids are correct, we then proceed to create a glycerol stock of the cell culture as well as a miniprep stock of the plasmid in order to conduct further experimentation in yeast.

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. 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. The purpose of the dropout media is to ensure that only cells that contain our plasmid survive as the recombinant plasmid allows cells to produce uracil, an essential amino acid. Without the recombinant plasmid, the cell would be fatally deprived of uracil which has been "knocked out" of the plating media. After two days, three colonies are swiped from the plate and added to an overnight culture of 2-3mL Selective Dropout Media C-Uracil and 2% Glucose then incubated for another two days at 30^oC. After another 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. This problematice as GFP output will become related to the number of plasmids as well as the stability of the various degron constructs which will likely invalidate any results.

Relative Stability Analyzed via Flow Cytometry

Flow cytometry is a high throughput method of analyzing cells for various optical outputs, namely fluorescnce. A flow cytometer is an analytical instrument in which cells that have been suspended in a solution are passed through a narrow channel in which fluorescence of invidual cells can be measured.

By utilizing Flow Cytometry, we can measure the amount of GFP output within cells from each degron construct. Based on where the Degron is inserted, we expected a different level of fluorescence. As such, we expected to see the highest GFP production in our Deg0 construct, as it only contains the Gal4-Protein-VP16 complex with no Degron inserted, hence being the most stable. We expected that Deg2 and Deg3 would have a lower GFP production than Deg0 but higher than Deg1 and Deg4. This rationale was based on the fact that the Deg1 and Deg4 have the Degron exposed, making it more likely to be degraded by ubiquitination than in Deg2 and Deg3 which has the Degron buried inside the Gal4-Protein-VP16 complex.

Mutagenesis through Error Prone Polymerase Chain Reactions (E-PCRs)

In order to validate our system as being capable of selecting more stable protein variants, we have to produce mutations in our protein of interest. Those mutations could potentially be beneficial and could carry a stabilizing affect on the protein of interest. Error-prone PCR utilizes DNA-polymerase's error-prone nature and further increases the likelyhood of mutations by manipulating the conditions in which, DNA-polymerase operates in, thereby causing the polymerase to create errors in DNA sequencing which in turn will create changes in the protein construct. Once, the DNA coding of our protein has been changed we can express the mutations and analyze those stabilizing or de-stabilizing affects they have on our protein. The possible mutations can then be analyzed using the degron system and more stable mutations can be easily seen then sequenced.

Selecting Stable Variants through Fluorescence Activated Cell Sorting (F.A.C.S.)

In order to validate our system as being capable of selecting more stable protein variants, we have to produce mutations in our protein of interest. Those mutations could potentially be beneficial and could carry a stabilizing affect on the protein of interest. Error-prone PCR utilizes DNA-polymerase's error-prone nature and further increases the likelyhood of mutations by manipulating the conditions in which, DNA-polymerase operates in, thereby causing the polymerase to create errors in DNA sequencing which in turn will create changes in the protein construct. Once, the DNA coding of our protein has been changed we can express the mutations and analyze those stabilizing or de-stabilizing affects they have on our protein. The possible mutations can then be analyzed using the degron system and more stable mutations can be easily seen through FACS (see below) then subsequently sequenced. As we sequence the various mutants, we are also looking for convergence in which the genetic sequence as well as the amino acid sequences converge on a single or a few mutations that lead to significantly higher fluorescence and therefore, higher stability.

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

The results for the no-protein of interest degron constructs matched expected results in which Deg0 had the highest expect stability which correlated to the highest fluorescence output. These expectations were validated by our experiment results in which cells with Deg0 exhibited the highest fluorescent levels. Our second expectations were that Deg2 and Deg3 would exhibit middle levels of fluorscence, lower than Deg0 but higher than Deg1 or deg4. Once again, these expectations were validated by our experimental results. The results for the no protein of interest degron constructs are as follow, cells containing our Deg0 protein construct exhibited the highest fluorescent followed by Deg2 and deg3 and finally by Deg1 and Deg4 both of which exhibited baseline levels (no protein, no degron construct PYE1 cells) of fluorescence.
In order, to validate the system as whole we must analyze the degron constructs with a specific well studied protein to analyze that protein's stability with our system compared to the protein stability measurements from current existing techniques. There were three variants of a protein each with varying stabilities that were quantified using circular dichroism and guanidium hydrogen chloride melts. The protein BINDI had the highest stability followed by BbpD04.3 and then BbpD04. If our system is accurate, cells containing the BINID-Degron construct would exhibit the highest fluorescence output followed by BbpD04.3 and BbpD04 would show the lowest fluorescence output. Since there are 5 possible degron construct for each of our 3 proteins of interest, all 15 data points would have to match our expectations. We expect that BINDI Deg0 would have the highest fluorescence of the all protein of interest Deg0 constructs followed by BbpD04.3 Deg0 and BbpD04 Deg0. Next, BINDI Deg2/3 would have middle levels of fluorescence followed by BbpD04.3 Deg2/3 and BbpD04 Deg2/3. Finally, BINDI Deg1/4, BbpD04.3 Deg1/4 and BbpD04 Deg1/4 would have the lowest levels of fluorescence if not baseline levels. The experimental results accquired through flow cytometry show a rough correlation to these expections********more experiment required***********

Mutagenesis Results and Mutant Variant Analysis

Future Plans

Examination of more proteins

Like any new up and coming technique, the degron system, will require further testing with a larger variety of well studied mutant variants of a single protein as well as a larger number of well studied proteins in general before the system can truly be accepted.
Our current plans for the future are too test a protein 33RM2 and its less stable variant 33CL1 both of which are bind to PD-1 (a negative t-cell regulator that prevents the recognition of tumorous cells by the immune system). Since, the stability of both these proteins are known and have been verified using other techniques such as thermal melts, they are very suitable candidates for testing using our degron system.

Further evolving more stable variants of existing proteins

Throughout this past summer, out team has been evolving more stable variants of BINDI through error-prone PCR and going forwards we will continue this process and continually analyze the mutants with flow cytometry and select cells that exhibit higher fluorescence with FACS. Furthermore, our technique could be applied...
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