Assessing Mutations In Beta-Globin Gene For Improved Protein Folding
Abstract:
Bibliography/Citations:
Works Cited
“Amino Acid Physical Properties.” Thermo Fisher Scientific, https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/amino-acid-physical-properties.html. Accessed 16 March 2024.
Choudhury, Hom. “Understanding a protein fold: The physics, chemistry, and biology of α-helical coiled coils.” Journal of Biological Chemistry, 3 March 2023, https://www.jbc.org/article/S0021-9258(23)00221-1/fulltext. Accessed 16 March 2024.
Dubochet, Jacques, et al. “Uncovering protein structure - PMC.” NCBI, 25 September 2020, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545034/. Accessed 16 March 2024.
“HBB - Hemoglobin subunit beta - Homo sapiens (Human) | UniProtKB.” UniProt, https://www.uniprot.org/uniprotkb/P68871/entry#structure. Accessed 16 March 2024.
“Hemoglobin subunit beta.” Wikipedia, https://en.wikipedia.org/wiki/Hemoglobin_subunit_beta. Accessed 16 March 2024.
“Hydrophobic-Hydrophilic Forces in Protein Folding - PMC.” NCBI, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604469/. Accessed 16 March 2024.
MA, Carlberg K. “Beta-Thalassemia - GeneReviews®.” NCBI, 8 February 2024, https://www.ncbi.nlm.nih.gov/books/NBK1426/. Accessed 16 March 2024.
“Sickle Cell Disease: A Review.” PubMed, 5 July 2022, https://pubmed.ncbi.nlm.nih.gov/35788790/. Accessed 16 March 2024.
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Additional Project Information
Research Plan:
A) Problem Being Addressed:
The problem being addressed is the prevalence of beta-Thalassemia, which is a disease caused by a mutation in the beta-Thalassemia gene, located on chromosome 11. Symptoms associated with this disease include extreme fatigue, shortness of breath, and affects the development of young children. The beta-Thalassemia gene is controlled by the beta-globin protein (subunit of the larger protein, hemoglobin).
B) Goals and Expected Outcomes
The goal of this project is to mutate the beta globin protein via pET15B (plasmid vector synthesized by GenScrip) and assess the changes made in protein production. We have used the 3D model of the beta-globin protein (as found on UniProt - #P68871) as a guideline to identify which amino acid residues are the most important for folding and protein production.
Increases/decreases in protein production are caused when a mutation changes an amino acid, and therefore its properties. For instance, non-polar amino acids are known for composing the core of a protein structure, whereas polar amino acids form the external frame. Therefore, a change in an amino acid from one type to another can cause instability. Furthermore, the bends and physical structure of a protein are also determined by the side chain interactions of amino acids. For example, hydrophobic amino acids form Van der Waal interactions, which are known to be weak and easily breakable. Polar amino acids, however, can form hydrogen bonds, which are stronger.
The most ideal outcome is if we can identify a mutation that both folds correctly (exhibits no change in comparison to the original 3D structure) and also increases protein production. Another positive outcome is if we can identify a mutation that folds correctly but decreases the protein production.
C) Procedure
- Receive a pET15B, a plasmid vector containing beta-Globin, synthesized by GenScript.
- Inoculate/transform 2 µL of plasmid DNA into the E. coli cells by plating it on Amp-resistant plates made of agar and letting it multiply by incubating at 37 degrees Celsius overnight.
- Isolate the purified plasmid DNA using the Zymo Pure Miniprep Kit (and its instructions).
- Run the Wild Type/WT DNA (unmutated plasmid DNA) miniprep product through gel electrophoresis.
- View the WT product through a trans-illuminator.
- Conduct research to determine the most ideal mutations to create a change in protein production, based on the properties of the amino acids being changed.
- Plan the single and double mutations (6 to 8 total mutations), find the forward and reverse primers, and calculate the annealing temperatures.
- Order primers from GenScript based on information found during the previous step.
- Conduct a Polymerase Chain Reaction(PCR) procedure (following the protocol from Accuris (TM) High Fidelity Master Mix) via a thermal cycler. This procedure is conducted to amplify the DNA.
- Run the PCR results through gel electrophoresis.
- View the PCR product through a trans-illuminator.
- Transform the mutated DNA into E.coli through same process mentioned in step 2.
- Send the mutated DNA to GenScript for sequencing (to confirm the mutations).
- Grow the mutated DNA into a 30 mL bacterial culture.
- Add IPTG (Isopropyl β- d-1-thiogalactopyranoside) to initiate the protein production process. IPTG is a chemical compound that plays a role similar to lactose, but does not get broken down by bacteria, which allows certain proteins to be expressed.
- Measure the optical density of the bacterial culture via a spectrophotometer. The ideal optical density (OD) to induce protein production in the culture is 0.8 (measured by the spectrophotometer readings).
- Run the induced culture products for various times in an SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). This gel serves the same purpose as an agarose gel, except that it is specially designed to measure the concentration of proteins, and not DNA.
- Start the protein purification of these mutant constructs using a Zymo Research 6x His (histadine) Tag Binding Column.
- Follow the protocol of the Zymo Pure His-Spin Protein Prep Procedure. This is done by transferring His-Affinity Gel into the His-Tag Binding Column, centrifuging it, washing it down with His-Wash Buffer, and eluting the product with the use of the His-Elution Buffer.
- Quantify the protein amount for both WT and mutants by using a spectrophotometer at 280 nm.
- Use a spectrophotometer to record the protein production of each mutant and of the WT.
- Compare the spectrophotometer readings on Excel (to prove or disprove hypothesis). Use error bars (determined by standard deviation and standard error of the mean) to evaluate findings.
D) Subject Specific Safety Risks
- Describe Biosafety Level Assessment process and resultant BSL determination
- The BSL Level for the Yard Sciences lab is BSL 1, because the project only involves plasmid, recombinant DNA, and dH5 Alpha E.coli competent cells. The aforementioned materials are all approved for a BSL 1 environment.
- Give source of agent, source of specific cell line, etc.
- The beta-globin gene was subcloned into a protein expression vector, pET15B, by the company GenScript.
- Detail safety precautions
- I have completed and been certified by the OSHA Safety Course.
- All biohazardous materials are autoclaved in a bag to fully sterilize them.
- A fume hood is located for the use of hazardous chemicals and in order to exhaust vapors/gases.
- Members of the lab wear PPE (Personal Protective Equipment), including lab coats and gloves.
- There is a Material Safety Data Sheet (MSDS) that contains instructions for using lab safety materials, and instructions for lab safety crises.
- All tables are cleaned regularly with an ethanol spray.
- Instead of using the potentially carcinogenic loading dye, Ethidium Bromide, during the PCR process, we use SybrGreen instead.
- Discuss methods of disposal
- All biohazard and chemical waste is collected in appropriate containers within the lab, and is disposed of by the company Choice Med Waste.
Questions and Answers
Initial Project Questions
1. What was the major objective of your project and what was your plan to achieve it?
The major objective of my project was to find mutations in the beta-globin gene that would either increase or decrease protein production. My plan to achieve it included: preparing a plasmid containing the protein, identifying mutations that could affect protein production, introducing these mutations and amplifying the DNA insert through PCR, inducing protein production in a 30 mL culture, and recording the differences in OD (via spectrophotometer) and in protein concentration (via SDS-PAGE).
a. Was that goal the result of any specific situation, experience, or problem you encountered?
My personal experience with a family member suffering from a disease inspired me to focus my project around medical sciences and disease investigation. Genetic disorders especially stood out to me, since they are not influenced by one’s lifestyle, but rather are predetermined. After conducting research on prevalent genetic diseases, I found that Sickle-Cell Anemia and Thalassemia were both caused by mutations in the beta-Globin gene. This was interesting to me, and I was curious as to what made this specific gene special. I read that Sickle-Cell Anemia already had many treatments in the works, and that Thalassemia was less explored. Therefore, I chose the latter as the subject for my project.
b. Were you trying to solve a problem, answer a question, or test a hypothesis?
I was trying to both solve a problem and test a hypothesis. Based on background research, I made an educated guess as to which mutations would affect protein production/folding. However, the inspiration for this hypothesis was derived from the need to solve the problem of Thalassemia’s prevalence.
2. What were the major tasks you had to perform in order to complete your project?
Some major tasks I had to perform in order to complete my project were:
- Transformation of the plasmid into E.coli cells and inoculation into Luria Broth
- Plasmid miniprep (the process through which the plasmid DNA is purified/isolated and the genomic DNA from the E.coli cells is removed)
- Making and running gels (helped measure the DNA)
- Researching (helped identify the most relevant mutations based on past studies)
- Conduct PCRs (and Gradient PCRs to assess ideal annealing temperatures)
- Measuring Optical Density (OD) on a spectrophotometer and recording the results
- Lysing cells via a MonoLyser
- Conducting a His-Tag Miniprep specially for protein purification
- Running a SDS-PAGE to quantify protein production
- Visually representing/comparing the results on a graph.
- Determining the connections between the results of the experiment with the properties of amino acids
a. For teams, describe what each member worked on.
The project was conducted individually, and therefore, this question does not apply.
3. What is new or novel about your project?
a. Is there some aspect of your project's objective, or how you achieved it that you haven't done before?
From the readings I have examined, many scientists have already used previous studies to assess beta-Globin protein expression. According to my research, such mutations include: frameshift mutations, deletions, and RNA splicing.
b. Is your project's objective, or the way you implemented it, different from anything you have seen?
No, there have been other scientists who have tried to identify mutations that cause beta-Thalassemia.
c. If you believe your work to be unique in some way, what research have you done to confirm that it is?
4. What was the most challenging part of completing your project?
The most challenging part of completing my project was definitely the Polymerase Chain Reaction (PCR) process. I originally attempted running all 13 PCR solutions at an annealing temperature of 63 deg C. However, the results were not strong for all solutions, and I realized that each one annealed best at a different temperature.
a. What problems did you encounter, and how did you overcome them?
To overcome the issue of finding the optimal annealing temperature for the PCR process, I had to conduct a gradient PCR. Instead of making one PCR solution per set of primers, I made six solutions. I set the thermal cycler settings so that each column would experience a different temperature. This way, each PCR solution had a version of it that was undergoing the process at a different annealing temperature. I then ran all 78 PCR solutions (6 solutions per each of the 13 mutants) on gels. The lanes with the strongest bands helped me identify which annealing temperature (if any) worked best for the specific mutant.
Another major problem I encountered was not having enough plasmid DNA presented in the gel electrophoresis that was run after the miniprep. This indicated that one of the following things were true:
- The Wild Type DNA was not strong enough
- There had been an error in the transformation/inoculation process
- There had been an error in the miniprep process
To overcome this issue, I had to redo the transformation and inoculation process multiple times. I tried transforming different amounts of the plasmid DNA to see which one would produce the best results.
Another problem I had was determining which mutations were the most important and would have the largest impact on protein production. I overcame this by relying on research of past studies to guide my selection. I also did deeper studies into the properties of amino acids and how they would affect the mutations.
b. What did you learn from overcoming these problems?
Conducting the gradient PCR taught me that trial and error isn’t always reliable. Sometimes, an orderly experiment is necessary. In this case, that meant setting up a scenario in which only the independent variable was changing (the temperature), and everything else was controlled (amount of PCR solution, number of PCR solutions per mutant etc.).
Overcoming the problem of unsuccessful gel electrophoresis taught me that the process of science heavily involves repetition. The desired outcomes do not always appear after the first try, and sometimes never do. I had to repeat the processes of inoculation, miniprep and running the gel three times in order to get the best results. This also taught me to be careful during inoculation by making sure I can visibly see the DNA enter the Luria Broth. I also learnt to double check the miniprep procedure to avoid pipetting any genomic DNA.
The problem of isolating the best mutations taught me how to conduct effective and meaningful research. I discovered how to find the most reliable sources, and how to gather information from previously conducted experiments to guide my own, while still maintaining an original idea.
5. If you were going to do this project again, are there any things you would do differently the next time?
If I were to redo this project, I would definitely run the gradient PCR more times so that I would have more mutants to conduct protein production on. This would have maybe even helped me identify an epitope.
Due to lack of time, I also did not wait for each protein to reach the OD of 0.8. If I had to redo the experiment, I would have checked the OD every two hours until every mutant had reached 0.8; since it is the optimal level for protein production.
With a second chance at this experiment, I would have also tested more mutations and maybe even tried frameshift mutations or deletions. I could have investigated the difference in protein production if the total number of amino acids had changed. The deletion aspect would have also related to Alpha-Thalassemia.
6. Did working on this project give you any ideas for other projects?
Yes, it did. Sickle cell disease is also caused by mutations in the Beta-globin gene, just like beta-Thalassemia. A future project could be conducted on which specific mutations affect sickle cell, and could even be compared to the ones that affect beta-Thalassemia and the results of my current project.
Working on this project also made me think about the different medications that are offered for patients with beta-Thalassemia. According to NIH, the drug luspatercept can be used to decrease the need for blood transfusions in a beta-Thalassemia patient. Maybe I could do a project relating to blood transfusions, and how certain factors can impact them, like blood typing etc.
7. How did COVID-19 affect the completion of your project?
COVID-19 did not have any major effect on the completion of my project. All of my work was completed at the YARD Sciences lab and at home, and there were no concerns about COVID-19 at either of these locations.