Ishika

===Hey everyone! My name is Ishika. I'm from Morgantown, West Virginia, and I am a rising senior at Morgantown High School. I have lived in Morgantown for the past 17 years. My interests include dancing, watching football (Steelers fan!), swimming, and figure skating. In addition, I am very interested in government and politics. I love traveling and volunteering.=== ===I was looking for summer programs online, and BLI immediately caught my attention. I interned at a company last summer, so I was looking for more of a research experience. My internship last summer focused on software engineering and computer science. While the internship was an extremely rewarding experience, I realized that my career interests were different. When I saw that BLI offered a biological research component to their program, I knew it would be an incredible opportunity.=== ===In the future, I plan to attend med-school to become a pediatrician, or law school to practice corporate law. Because my interests are broad, I plan on majoring in biology and minoring in political science in college. I hope that by taking classes in both areas, I will be able to hone my interests.===

Come talk to me if you want to know more! I'm looking forward to a great three weeks!
Research Project: Rewriting the Genetic Code DNA consists of sequences of nucleotides that code for 64 distinct codons. These nucleotides (A, C, T, and G), in groups of three, form amino acids for the most part. However, a few of these codons tell a cell when to stop adding amino acids to the protein chain. These codons, called “stop” codons, have been the basis of research into rewriting the genetic code.

Redesigned genomes require advances that merge the desired biological behavior with challenges integral to biological complexity. Recently, researchers from MIT and Harvard have developed technologies that allow them to alter the genetic code, and ultimately replace particular DNA sequences throughout the genome of a living cell. These researchers concentrated on a specific stop codon, consisting of the letters TAG. Seeing as the TAG stop codon is the most rare in the E. coli genome, it was an ideal choice to begin researching.

The researchers used two techniques to replace this stop codon. Multiplex Automated Genome Engineering was used to locate specific DNA sequences and replace them with a new sequence of nucleotides. Simultaneously, the cell replicated its DNA. This was so that the stop codons being targeted could be changed, while the rest of the genome remains the same. The research conducted replaced the TAG codon with a different stop codon, TAA. The E. coli genetic code contains three stop codons (UAG, UAA, and UGA) whose translation termination is facilitated by two release factors, RF1 and RF2. RF1 recognizes the stop codons UAA and UAG, whil RF2 recognizes UAA and UGA. It was hypothesized that replacing all TAG codons with TAA codons would end genetic dependence on the RF1 release factor. This would allow the new stop codons to be recognized by RF2. The researchers used MAGE to engineer 32 strains of E. coli, each of which consisted of 10 TAG codons that were replaced. The ecNR2 genome was divided into 32 regions; 31 of which contained 10 targets, and the other containing the remaining four targets.

After creating these strains, researchers realized they would need to combine these strains to create one that contains all 314 edits. To perform this action, Conjugative Assembly Genome Engineering (CAGE) was created. CAGE uses bacterium to build an extension to a neighboring cell (recipient), and then uses that pathway to pass on the TAA codons.



In the figure above, the CAGE process is demonstrated. As you can see, each strain shared its DNA with another strain. After one round of CAGE, 16 strains had been produced that had double the number of TAG replacements that it started with. Then, those 16 strains underwent a second round of CAGE, producing 8 more strains. Once all of the TAG stop codons were deleted, the researchers’ next step was to delete the cell machinery that reads the TAG codon. Once this was done, they were free to create a new purpose for the DNA, such as encode a new amino acid.

By altering the genetic code, scientists could also engineer bacteria that are resistant to multiple viruses. In industries that cultivate bacteria, including pharmaceuticals and energy, viruses affect up to 20 percent of cultures, with a huge impact on productivity. However, those viruses can only infect a cell if the bacterial and viral genetic codes are the same.

These new technologies are a huge step towards the goal of replacing the expression of a specific codon. Such technology could enable scientists to design cells that build proteins not found in nature. Because the alterations were done in living cells, the researchers have been able to monitor any potential harmful effects as they appear. As of now, this method of rewriting the genetic code hasn’t disrupted the cells’ function. Preliminary characterization suggests that the altered bacteria still behave normally, and can survive and reproduce.

Design Project: Medulloblastoma Medulloblastoma is a highly malignant brain tumor in children, responsible for 25% of all pediatric brain cancers. Usually diagnosed from age 3 to age 8, medulloblastoma arises in the posterior fossa region, or cerebellum of the brain. Tumors grow quickly and can invade neighboring portions of the brain. If cancerous tumor cells get into the cerebrospinal fluid (CSF), medulloblastoma as well as the cancer can spread to other areas of the central nervous system. It is a highly invasive embryonal neuroepithetlial tumor that has the tendency to disseminate throughout the central nervous system early in its course. Medulloblastoma affects both sexes, but it is more common in boys.

Medulloblastomas generally appear in the vicinity of the fourth ventricle, between the brainstem and the cerebellum. Although it is thought that medulloblastomas originate from immature or embryonic cells at their earliest stage of development, the exact cell of origin, or “medulloblast” has yet to be identified. Recent research has shown that medulloblastoma arises from cerebellar stem cells that haven’t been able to differentiate into their normal cell types.

A recurrent mutation in the gene β-catenin, officially known as CTNNB1, was identified as cancer causing within the tumor. CTNNB1 is a proto-oncogene, simply meaning that it potential to cause cancer when expressed at increased levels. Most mutations of this gene cluster on a tiny area of the N-terminal segment of β-catenin, the β-TrCP binding motif. Loss-of-function mutations of this motif essentially make the degradation of β-catenin impossible.

With intensive surgery, radiotherapy, and chemotherapy, approximately 50% of children with medulloblastoma can be expected to be cancer-free 5 years later. The aim of the surgery is to remove as much tumor as possible. When modern techniques are used, most tumors can be completely removed. After treatment, tests are conducted to determine whether there is any residual tumor at the operated site. The chemotherapeutic regimens for medulloblastoma involve a combination of lomustine, cisplatin, carboplatin, vincristine, or cyclophosphamide. These chemotherapy drugs have the ability to treat other types of cancer as well. However, the tumor and the cancer have the ability to spread to other areas of the brain and spinal cord. In addition, these methods generally result in a considerable amount of damage to healthy tissue. Oncologists and researchers have been looking into cancer treatments that precisely distinguish between healthy and diseased tissue/cells.

My design proposes an alternate form of treatment to eliminate the cancer in medulloblastomas. Genetically engineered E. coli will be delivered to the tumor intravenously via the tumorigenic pathway for medulloblastoma. Within the E. coli, small hairpin RNA (shRNA) will be generated via RNA interference (RNAi). In order to do this, a bacterial expression vector must be present within the E. coli. In this case, we must use the pENTR expression vector. Once the expression vector has integrated into the E. coli, the shRNA will be transcribed by a polymerase II promoter. This product mimics pri-microRNA (pri-miRNA) and is processed by Drosha, a Class 2 RNase III enzyme responsible for initiating the following process. The E. coli will also be engineered to produce the invasin adhesion protein so that it is able to invade the cancerous cells. The invasin protein originates from Yersinia pseudotuberculosis. It mediates the entry into cancerous cells by binding several β1 chain integrin receptors. These receptors will bind to the fibronectin receptor α 5 β1 and immobilize on a filter membrane.

The resulting pre-shRNA will be exported from the E. coli by Exportin 5, a protein encoded by the XPO5 gene that enables the export of shRNA. This product is then processed by Dicer, an enzyme endoribonuclease in the RNase III family that cleaves double-stranded RNA (dsRNA) and pre-microRNA (miRNA) into short double-stranded RNA fragments, and loaded into the RNA-induced silencing complex (RISC). Using the adhesive proteins (invasin), the bacteria will be able to bind to the β1 integrin receptors on the cancerous cell and invade, inducing uptake. The shRNA segments will bind to CTNNB1 mRNA transcripts within the cell, which will ultimately induce mRNA cleavage and silence the CTNNB1 gene.

This design is far more advantageous than the current methods used to remove the cancer from medulloblastoma such as chemotherapy and radiation. It is commonly known that drugs affect “younger” tumors more effectively because mechanisms regulating cell growth are usually still preserved. With succeeding generations of tumor cells, differentiation is generally lost, growth becomes less regulated, and the tumors become less responsive to most chemotherapeutic agents. Often times, cell division in medulloblastomas effectively ceases, making them insensitive to chemotherapy. In addition, cancer cells have the ability to become more resistant to chemotherapy treatments over time. Using genetically engineered E. coli, cancer cells can be easily and effectively eliminated.

Bibliography:

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