Anna



Hi I'm Anna, I'm 15 and I'm from Austin, Texas. I attend school at the Liberal Arts and Science Academy and I'm a rising sophomore. I was born in Russia and spent the majority of my childhood moving back and forth between there and the states. Back home I have a crazy orange cat named Sonny and a lovable bear-looking dog named Ollie. I love biology, playing the accordion, shooting my bow at practice and tournaments, and fencing. At school I participate in Science Olympiad and Science Bowl. Biology and medicine have been my passion ever since I was very little. I'd also like to travel around the world and be part of addressing medical and public health care in developing countries as well as approaching new technologies for cellular work.

=** __Research Project: Quorum Sensing and Biofilms__ **=

Quorum sensing, a recent and vital discovery in the world of bacteria, is a system of inter and intra communication between bacteria that allow bacterial populations to collectively carry out gene expression based on signaling and response from each other and their environment. The system is coined “quorum sensing” because the bacteria are able to sense the accumulation of released molecules from neighboring organisms and determine how many cells are present around them. Afterwards they can simultaneously determine if the cell density is substantial to carry out the release of the wanted protein. The vibrio fischeri bacteria that are found in the body system of squids are gram negative and use this basic signaling system.

A single bacterium has a genetic sequence that codes for an autoinducer, or a signaling molecule that will be released into the environment. This molecule can vary for different types of bacteria. Gram negative bacteria, which are typically bacilli and have a thin cell wall use acyl-homoserine lactones as a signaling molecule (AHL). They diffuse easily in and out of a bacterial cells where they then bind to matching AHL receptors on the surfaces of other bacteria. Once the molecule reaches a certain threshold concentration it binds to and activates a regulatory protein. The regulatory protein then binds to a specific site on the DNA where it initiates the sequence to create a quorum sensing specific protein as well as more enzymes to make the AHL molecules necessary to continue the process.



In gram positive bacteria the autoinducer are precursor oligopeptides.The oligopeptides are cleaved into functional signaling molecules of 10-20 amino acids. Unlike AHLs, these autoinducers cannot easily diffuse across the membrane of the bacteria and must be exported via active transport proteins. When the amount of oligopeptides autoinducers reach a certain threshold amount outside of the cell, they are detected by sensors on the outer membrane of the cell. When the oligopeptide reacts with the sensor protein, the intracellular part of the sensor becomes phosphorylated. The phosphate is then transferred to a response regulator protein. The response regulator protein then allows to bind to a specific site on the DNA. It becomes a transcription factor as it alters the transcription of target genes. In this way quorum dependent proteins such as virulence factors are produced.



This is the intra communication system that is used by like bacteria to establish presence of their own kind. These molecules are released and reciprocated continuously. Each bacteria is able to send out a signals and receive one as well. This allows each individual bacteria to count how many other bacteria there are. Once the bacterium can assess that there is a proper amount of other bacteria present, it can simultaneously, along with the other bacteria emit a response such as virulence or bioluminescence

However, there is also the intra species communication system. The universal molecule used in interspecies communication is called autoinducer-2 or AI-2. The enzyme LuxS is responsible for creating the Al-2 molecule. The gene encoding the LuxS enzyme has been detected in 35 of 89 sequenced bacterial genomes with little variation. The LuxS enzyme uses the substrate dihydroxy pentanedione to create the AI-2 molecule.

Quorum sensing plays an important role in biofilms. Biofilms are groups of microorganisms that stick to each other and are frequently embedded in extracellular DNA, proteins and polysaccharides. The films can appear on living or nonliving surfaces, such as a table or someone’s gums. Formation occurs due to a number of factors including cellular recognition of specific or non-specific attachment sites on a surface or nutritional cues. When a cell starts growing in a biofilm, many genes are regulated differently.



Biofilms are a major concern for the food and health industry. Human pathogens form biofilms of food and food contact surfaces thereby enhancing their ability to survive harsh environments, resists antimicrobacterial treatments, and persist in the food processing environment.

The increases cell density environment of the biofilms favors chemical signals to communicate for social interactions. Quorum sensing is used to regulate biofilm formation and its maintenance as well as symbiosis between species of the biofilm. In 1998, Greenberg and his colleagues first described quorum sensing in Psuedomonas aeruginosa, a gram negative bacteria known for causing fatal systematic disease under certain conditions. Lung infections with biofilms including this pathogen are found in patients with <span style="background-color: transparent !important; background-position: initial initial !important; background-repeat: initial initial !important; border: none !important; display: inline !important; float: none !important; font-family: arial,helvetica,sans-serif !important; font-size: 13px !important; font-style: normal !important; font-variant: normal !important; font-weight: normal !important; height: auto !important; line-height: 19px !important; margin: 0px !important; min-height: 0px !important; min-width: 0px !important; padding: 0px !important; text-decoration: underline !important; vertical-align: baseline !important; width: auto !important;">. Subsequent studies further show that the quorum-sensing circuits in P. aeruginosaorchestrate a symphony of several virulence factors.

In Gram-positve bacteria, many virulence factors involved in staphylococcal infections, including surface-associated adhesins, hemolysin, toxins and autolysins, are regulated by quorum sensing via the accessory gene regulator (agr) system.

Knowledge of the chemical structures of different types of signaling molecules allows the identification of compounds that can be used to modulate quorum sensing-related processes, including biofilm formation. Additional research is needed to understand how quorum sensing works mechanistically in biofilms and how cell-to-cell signaling may influence the virulence and antimicrobial resistance of biofilm communities. This information is important to identify possible targets and to design strategies that control biofilm formation on industrial, medical, and food and food processing surfaces.

Biotechnolgists are looking into mutating the systems/proteins that produce the signaling molecules in quorum sensing in order to disrupt the biofilm and virulence.

__Observed and Studied Quorum Sensing in Biofilms Examples__ // ** Campylobacter jejuni ** //  spiral or spirochete, rod-shaped Gram-negative bacteria that can cause gastroenteritis and is commonly associated with foodborne illness. The production of AHLs has not been established in // C. // jejuni The enzyme LuxS was found in C. jejuni, and an AI-2-like product has been demonstrated. However, chemical characterization of these AI-2-like compounds has not been done. Utilizing the M129 strain of C. jejuni, scientists demonstrated that a mutation in the luxS gene led to a decrease in biofilm formation when compared to the isogenic wild-type strain. Addition from the wild-type strain M129 led to an increase in biofilm formation.

//A. hydrophila //is a Gram-negative rod and facultative anaerobe present in all freshwater environments and in brackish water. Some strains of //A. hydrophila //are capable of causing illness in fish and amphibians as well as in humans, who could acquire infections through open wounds or by ingestion of a sufficient number of the organisms in food or water. //A. hydrophila //also causes infections. The formation of mature biofilms on stainless steel coupons by // A. hydrophila //required the production of C4-HSL since an ahyI (AHL synthase) mutant lacking the ability to form C4-HSL did not produce a mature biofilm. A mutation in the ahyR (AHL receptor) gene had no effect on biofilm formation. An unidentified LuxR-like receptor bound the AHL to produce biofilm in the ahyR mutant. Thus, quorum sensing regulates biofilm formation, and a number of researchers have shown that mutations in flagella formation decrease biofilm formation in A. hydrophila. Although studies have not been reported, it is probable that flagella formation in //A. hydrophila //is regulated via quorum sensing.
 * // Aeromonas hydrophila // **

//B. cereus //is a rod-shaped facultative aerobic Gram-positive bacterium that forms endospores and has been associated with foodborne illness. The quorum sensing system of //B. cereus //<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; font-size: 12px;">consists of PlcR (the plcR gene encodes a transcriptional regulator) and PapR (the papR gene encodes a cell-to-cell signaling peptide) as well as the LuxS/AI-2 system A plcR-negative mutant produces approximately 4-fold more biofilm than its isogenic wild-type parent.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; font-size: 12px; vertical-align: baseline;">// Bacillus cereus // **

<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; font-size: 12px; vertical-align: baseline;">// ** Listeria monocytogenes ** // <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; font-size: 12px; vertical-align: baseline;">// L. monocytogenes //is a facultative pathogenic Gram-positive coccoid rod-shaped bacterium and the cause of listeriosis. It is associated with foods such as milk, cheeses, ice cream, raw vegetables, fermented raw-meat sausages, raw and ready-to-eat meat and poultry, and raw and smoked fish. Resistant to the injurious effects of freezing, drying, and heating, it is able to grow at temperatures as low as 3 °C. Biofilm formation by // L. monocytogenes //has been demonstrated on polyvinyl chloride plates, glass slides, stainless steel, polyethylene, teflon coupons, conveyer belt materials (such as polypropylene, acetal, stainless steel), and floor drains of food processing facilities.

<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; font-size: 12px; vertical-align: baseline;">While// L. monocytogenes //has a LuxS/AI-2 system, the luxS gene was shown to repress biofilm formation A mutation in the luxS gene resulted in a 4-fold thicker biofilm than in the wild type, and the addition of in vitro synthesized AI-2 to cultures of the mutant did not repress biofilm formation. Therefore, there is no indication that there is a quorum sensing role for AI-2 in // L. monocytogenes //biofilm formation.

<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; font-size: 12px; vertical-align: baseline;">Little is known about possible peptide quorum sensing compounds in// L. monocytogenes //. However, recent data indicate that // L. monocytogenes //has an accessory gene regulator (agr) system. There are 4 genes, agrB, agrD, agrC, and agrA, in the agr operon. There is an approximately 62% decrease in the number of cells attached to glass slides with agrA and agrD deletion mutants of L. monocytogenes as compared to the wild type. In addition, these mutants showed a 33% decrease in the amount of biofilm formed on polystyrene during the first 24 hours. However, at 48 and 72 h, the amount of biofilm formed by the mutants and wild type was approximately the same.

<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; font-size: 12px; vertical-align: baseline;">// ** Escherichia coli ** // <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; font-size: 12px; vertical-align: baseline;">// E. coli //is a facultative anaerobic Gram-negative rod-shaped bacterium that causes gastrointestinal and extra-intestinal infections. // E. coli //can also cause hemorrhagic colitis and hemolytic uremic syndrome, and outbreaks have been linked to contaminated water and foods such as ground beef, raw milk, and produce. // E. coli //strains produce biofilms on the surfaces of glass, stainless steel, etc. The LuxS/AI-2 system is present in E. coli Enzymatically synthesized AI-2 increased motility and stimulated the formation of biofilms when added to wild-type // E. coli //K-12 strains

<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; font-size: 12px; vertical-align: baseline;">// ** Vibrio cholerae ** // <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; font-size: 12px; vertical-align: baseline;">Members of the genus Vibrio are facultative anaerobic Gram-negative curved rod-shaped bacteria that are associated with foodborne and waterborne diseases.// V. cholerae //causes cholera whereas V. vulnificus is associated with wound infections, enteritis, bacteremia, and death in immunocompromised individuals. A number of studies have examined quorum sensing in vibrios. As a wound pathogen, the organism may form biofilms in human tissue. <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; font-size: 12px; vertical-align: baseline;">Biofilm formation in // V. cholerae //is tightly regulated and controlled by multiple quorum sensing systems operating simultaneously to regulate the transcription of genes involved in the production of exopolysaccharide. The organism forms biofilms at low (rather than high) cell densities when signal molecules have accumulated. Furthermore, exopolysaccharide-overproducing variants readily arose during the time course of the biofilm assay, trapping smooth parental cells within the biofilm. Mutations in hapR, a transcriptional regulator, were responsible for enhanced biofilm formation. This suggests that at low cell densities or early in the infection, it may be advantageous for // V. cholerae //to be able to form biofilms and express virulence genes.

Studying the varied mechanisms of quorum sensing in biofilms allows scientists to develop systems for disrupting the mechanism and preventing illness and infection.

=** __Design__ __ Project: Ep-CAM gene silencing in Craniopharyngiomas __**= Craniopharyngiomas are rare benign brain tumors that account for 5-16% of all intracranial child tumors. They originate from the pituitary stalk and grow out into the areas surrounding it including: the pituitary gland, hypothalamus, and optic tract. These tumors are mostly present in children between the ages of 5 and fourteen but also in adults between the ages of 50 and 70. The tumors are slow growing in most cases and can grow several years without being detected. They can by cystic (pouch that contains fluid), or solid with proteins and calcium components. Diagnosis of the condition is done based on a number of endocrine and visual disturbances that only become apparent once the tumor has become at least 3 cm in diameter and has begun to heavily afflict and compress on the surrounding vital centers.
 * __ Background: __**

While benign and immobile, the tumors press onto the vital structures as it develops on and around, damaging their function. Depending on where the tumor is located, craniopharyngiomas cause dangerous endocrine dysfunctions that can lead to eventual death even with partial or complete removal of the tumor. If the tumor presses on the pituitary gland or the pituitary stalk, it can cause stunted growth (especially in children), delayed puberty, fatigue, low blood pressure, depression, and diabetes insipidus (where the kidneys are unable to conserve water). When the tumor presses on the optic tract, it can cause blurred vision or complete blindness. If the tumor presses on the hypothalamus of the brain, obesity, headaches, and body temperature regulation abnormalities can occur.

Craniophayrngiomas are particularly difficult to remove because of their strong adhesive nature to the surrounding vital structures. The transition zone between the glial tissue and tumor cells consists of an irregular and complex protein border that makes it hard to identify a cleavage line to remove the tumor from the tissues. Total removal of the tumor could then damage the vital parts of the brain further. As a result most surgeries leave part of the tumor tissue behind.

Post-operative survival rates are high but reoccurrence of the neoplastic growth from the remnants of the surgery is common. After surgery, the damage on the glial tissue still remains and endocrine and visual functions of damaged centers persevere. The reoccurrence of the tumor only worsens these already existing conditions. Most of mortality from craniopharyngiomas, especially in children, occurs after the reoccurrence of the tumor.

In recent studies, the Ep-CAM gene that codes for cell adhesion glycoproteins has been linked to the aggressive adhesive properties and reoccurrence of craniopharyngiomas. The expression of this gene is present in over 60% of reoccurring craniopharyngiomas. The gene is also associated with other cancers in tumorigenesis and metastasis.

Radiotherapy is commonly utilized after surgery to help remove remnants of the brain tumor that could not be resected with surgery. High energy radiation is used to shrink tumors and kill cancer cells. X-rays, gamma rays, and charged particles are used for this treatment. Radioactive iodine travels in the blood to kill cancer cells. The therapy destroys cancer cells by damaging their DNA directly or creating charged particles within the cells that will then kill the cancer cells.
 * __ Competing Technologies __**

However, often the radiotherapy is damaging to the remaining healthy tissues of the brain. Damage to the rapidly dividing cells of normal tissues can result in loss of hair, damage to the salivary glands, and damage to the hypothalamus. Later effects of radiation therapy can include: replacement of normal tissue with scar tissue (leading to restricted movement in the afflicted area), damage to internal organs resulting in diarrhea or bleeding, memory los, infertility, and sometimes a second cancer from the radiation particles themselves. The risk of developing a second cancer is greatly increased in children and adolescents. Such side effects lead to controversy on using radiotherapy in order to remove the remaining craniopharyngioma cancer cells.

The design focuses on silencing the Ep-CAM gene within the tumor center for two purposes: decreasing strength of cell-to-cell connection within the neoplastic growth and cell to glial tissue connection of the surrounding structures. Recent studies show that methylation of CpG islands on the Ep-CAM promoter sequence will silence the gene. The CpG islands are cites where are there is a high density of Guanine and Cytosine amine acids along with their phosphorous bonds.
 * __ Design __**

The aerotolerant bacteria would enter the body intravenously through the tumorigenic pathway. The bacteria will be engineered with an Ep-CAM promoter specific m5c DNA methyltransferase gene to produce enzymes that will attach the extra methyl group (CH3 molecule) to the CpG islands. The bacteria are genetically engineered with sensors for the Ep-CAM glycoprotein and calcified material, an abnormal characteristic in soft tissue and present homogeneously throughout craniopharyngioma growths. The E. coli will also be engineered to produce the invasin adhesion protein so that it is able to invade the cancerous cells. It mediates the entry into cancerous cells by binding several β1 chain integrin receptors.

When the bacteria reach the tumor growth inside the brain, they will respond to the presence of calcified material and the Ep-CAM glycoprotein by synthesizing the m5c DNA methyltransferase enzymes. They will then enter the tumor cells using the invasin proteins binding to the B-1 integrin receptors. Once they enter the cells, the m5c DNA methyltransferase enzymes will methylate the CpG islands of the Ep-CAM promoter region. The RNA Polymerase will not be able to bind and Ep-CAM expression is silenced.
 * __Logic Gates and Truth Tables__**

//AND Logic Gate// In an AND gate, both inputs must be present in order to produce the output. In this case both inputs are needed in order to ensure that the bacteria release the product into the tumor growth and not in an unintended area of the brain.
 * Ep-CAM glycoprotein || Calcification Deposits || m5c methyltransferase enzymes ||
 * 0 || 0 || 0 ||
 * 1 || 0 || 0 ||
 * 0 || 1 || 0 ||
 * 1 || 1 || 1 ||

//Inverter (NOT) Gate// In an inverter or NOT gate, the input must not be present in orderfor the output to be produced. However in this case, the output is not desired and the presence of the input is essential.
 * M5c methylation of CpG Islands on Pomoter || Ep-CAM Expression ||
 * 0 || 1 ||
 * 1 || 0 ||

Ideally, the design would be much less detrimental that radiotherapy. The use of bacteria cells has already been investigated in invading cancer cells and completely destroying them with little to no harmful side effects shown thus far. The design would cause the tumor and its adherence to surrounding structures to be a lot weaker. This would hopefully aid surgeons to remove more of the tumor on the first occurrence of the tumor. Also once the tumor has been removed, the design would helpfully prevent remnants from growing back, and craniopharyngioma from reoccurring once more. Then less or no radiotherapy will be required to remove the tumor remnants.
 * __ Advantages of the Design __**

The design is complex and would require careful consideration of each step, especially making it so that the methylation enzymes specifically target the promoter region and no other part of the cell or any surrounding healthy tissues.
 * __ Possible Problems: __**

There have been many considerations and doubts in the synthetic biology field and letting bacterial agents enter food supply or mammalialian cells. However, studies have already been done on engineering bacterial cells to invade and destroy cancer cells and in the production of artemisinin, thus this similar design should also be valued at the same merit.

Working with the invasin protein will require laboratory and clinical precaution as invading healthy cells is something to avoid. While there could always be some kind of risk associated when working with bacteria, the proposed rewards outweigh the risks. The system could possible alleviate many of the worst symptoms of craniopharyngiomas and prevent reoccurrence of the tumor after primary removal.

The design would first be detested on tissue culture and then move on to animal testing. If the design proves to be successful in all stages, including any further human tests, a similar construct could be applied and engineered to other molecules that are linked to tumorigenesis. Of course the aspects of delivering the bacteria would have to be localized depending on where the tumor is located.
 * __ Testing __**


 * __Bibliography__**

<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">"Quorum Sensing in Biofilms: Why Bacteria Behave the Way They Do." //<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">- IFT.org //<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">. N.p., n.d. Web. 01 Aug. 2013.

<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">Li, Yung-Hua, and Xiaolin Tian. "Abstract." //<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">National Center for Biotechnology Information //<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">. U.S. National Library of Medicine, 23 Feb. 2012. Web. 01 Aug. 2013.

<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">Lasky, Joseph L., III. "Pediatric Craniopharyngioma ." //<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">Pediatric Craniopharyngioma //<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">. N.p., n.d. Web. 01 Aug. 2013.

<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">Gun, Bernardina. "EP-CAM in Carcinogenesis." , University Medical Centre Groningen, Hanzeplein, 13 Sept. 2010. Web. 31 July 2013.

<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">Shiah, Shine-Gwo. "Epigenetic Regulation of EpCAM in TumorInvasion and Metastasis." Nation Institute of Cancer Research, n.d. Web. 01 Aug. 2013.

<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">Karavitaki, Niki. "Endocrine Reviews." //<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">Craniopharyngiomas //<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">. Endocrine Reviews, 2006. Web. 01 Aug. 2013.

<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">"Craniopharyngioma." Barrow Neurological Institute, n.d. Web. 01 Aug. 2013.

<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">"Radiation Therapy." //<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">Radiation Therapy for Cancer - //<span style="background-color: #ffffff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">. National Cancer Institute, n.d. Web. 01 Aug. 2013