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Detection of Gut Inflammation and Tumors Using Photoacoustic Imaging


Detection of Gut Inflammation and Tumors Using Photoacoustic Imaging


Photoacoustic imaging is a technique in which contrast agents absorb photon energy and emit signals that can be analyzed by ultrasound transducers. This method allows for unprecedented depth imaging that can provide a non-invasive alternative to current diagnostic tools used to detect internal tissue inflammation.1 The Rice iGEM team strove to use photoacoustic technology and biomarkers to develop a noninvasive method of locally detecting gut inflammation and colon cancer. As a first step, we genetically engineered Escherichia coli to express near-infrared fluorescent proteins iRFP670 and iRFP713 and conducted tests using biomarkers to determine whether expression was confined to a singular local area.


In photoacoustic imaging, laser pulses of a specific, predetermined wavelength (the excitation wavelength) activate and thermally excite a contrast agent such as a pigment or protein. The heat makes the contrast agent contract and expand producing an ultrasonic emission wavelength longer than the excitation wavelength used. The emission wavelength data are used to produce 2D or 3D images of tissues that have high resolution and contrast.2

The objective of this photoacoustic imaging project is to engineer bacteria to produce contrast agents in the presence of biomarkers specific to gut inflammation and colon cancer and ultimately to deliver the bacteria into the intestines. The bacteria will produce the contrast agents in response to certain biomarkers and lasers will excite the contrast agents, which will emit signals in local, targeted areas, allowing for a non-invasive imaging method. Our goal is to develop a non-invasive photoacoustic imaging delivery method that uses engineered bacteria to report gut inflammation and identify colon cancer. To achieve this, we constructed plasmids that have a nitric-oxide-sensing promoter (soxR/S) or a hypoxia-sensing promoter (narK or fdhf) fused to genes encoding near-infrared fluorescent proteins or violacein with emission wavelengths of 670 nm (iRFP670) and 713 nm (iRFP713). Nitric oxide and hypoxia, biological markers of gut inflammation in both mice and humans, would therefore promote expression of the desired iRFPs or violacein.3,4

Results and Discussion


To test the inducibility and detectability of our iRFPs, we used pBAD, a promoter that is part of the arabinose operon located in E. coli.5 We formed genetic circuits consisting of the pBAD expression system and iRFP670 and iRFP713 (Fig. 1a). AraC, a constitutively produced transcription regulator, changes form in the presence of arabinose sugar, allowing for the activation of the pBAD promoter.

CT Figure 1b.jpg

Fluorescence levels emitted by the iRFPs increased significantly when placed in wells containing increasing concentrations of arabinose (Figure 2). This correlation suggests that our selected iRFPs fluoresce sufficiently when promoters are induced by environmental signals. The results of the arabinose assays showed that we successfully produced iRFPs; the next steps were to engineer bacteria to produce the same iRFPs under nitric oxide and hypoxia.

Nitric Oxide

The next step was to test the nitric oxide induction of iRFP fluorescence. We used a genetic circuit consisting of a constitutive promoter and the soxR gene, which in turn expresses the SoxR protein (Figure 1b). In the presence of nitric oxide, SoxR changes form to activate the promoter soxS, which activates the expression of the desired gene. The source of nitric oxide added to our engineered bacteria samples was diethylenetriamine/nitric oxide adduct (DETA/NO).

Figure 3 shows no significant difference of fluorescence/OD600 between DETA/NO concentrations. This finding implies that our engineered bacteria were unable to detect the nitric oxide biomarker and produce iRFP; future troubleshooting includes verifying promoter strength and correct sample conditions. Furthermore, nitric oxide has an extremely short half-life of a few seconds, which may not be enough time for most of the engineered bacteria to sense the nitric oxide, limiting iRFP production and fluorescence.

CT Figure 1c.jpg


We also tested the induction of iRFP fluorescence with the hypoxia-inducible promoters narK and fdhf. We expected iRFP production and fluorescence to increase when using the narK and fdhf promoters in anaerobic conditions (Figure 1c and d).

However, we observed the opposite result. A decreased fluorescence for both iRFP constructs in both promoters was measured when exposed to hypoxia (Figure 4). This finding suggests that our engineered bacteria were unable to detect the hypoxia biomarker and produce iRFP; future troubleshooting includes verifying promoter strength and correct sample conditions.

Future Directions

Further studies include testing the engineered bacteria co-cultured with colon cancer cells and developing other constructs that will enable bacteria to sense carcinogenic tumors and make them fluoresce for imaging and treatment purposes.

Violacein has anti-cancer therapy potential

Violacein is a fluorescent pigment for in vivo photoacoustic imaging in the near-infrared range and shows anti-tumoral activity6. It has high potential for future work in bacterial tumor targeting. We have succeeded in constructing violacein using Golden Gate shuffling7 and intend to use it in experiments such as the nitric oxide and hypoxia assays we used for iRFP670 and 713.

Invasin can allow for targeted cell therapy

Using a beta integrin called invasin, certain bacteria are able to invade mammalian cells.8-9 If we engineer E. coli that have the beta integrin invasion as well as the genetic circuits capable of sensing nitric oxide and/or hypoxia, we can potentially allow the E. coli to invade colon cells and release contrast agents for photoacoustic imaging or therapeutic agents such as violacein only in the presence of specific biomarkers.10 Additionally, if we engineer the bacteria that exhibit invasin to invade colon cancer cells only and not normal cells, then this approach would potentially allow for a localized targeting and treatment of cancerous tumors. This design allows us to create scenarios with parameters more similar to the conditions observed in the human gut as we will be unable to test our engineered bacteria in an actual human gut.


The International Genetically Engineered Machine (iGEM) Foundation ( is an independent, non-profit organization dedicated to education and competition, the advancement of synthetic biology, and the development of an open community and collaboration.

This project would not have been possible without the patient instruction and generous encouragement of our Principal Investigators (Dr. Beth Beason-Abmayr and Dr. Jonathan Silberg, BioSciences at Rice), our graduate student advisors and our undergraduate team. I would also like to thank our iGEM collaborators.

This work was supported by the Wiess School of Natural Sciences and the George R. Brown School of Engineering and the Departments of BioSciences, Bioengineering, and Chemical and Biomolecular Engineering at Rice University; Dr. Rebecca Richards-Kortum, HHMI Pre-College and Undergraduate Science Education Program Grant #52008107; and Dr. George N. Phillips, Jr., Looney Endowment Fund.

If you would like to know more information about our project and our team, please visit our iGEM wiki at


  1. Ntziachristos, V. Nat Methods. 2010, 7, 603-614.
  2. Weber, J. et al. Nat Methods. 2016, 13, 639-650.
  3. Archer, E. J. et al. ACS Synth. Biol. 2012, 1, 451–457.
  4. Hӧckel, M.; Vaupel, P. JNCI J Natl Cancer Inst. 2001, 93, 266−276.
  5. Guzman, L. M. et al. J of Bacteriology. 1995, 177, 4121-4130.
  6. Shcherbakova, D. M.; Verkhusha, V. V. Nat Methods. 2013, 10, 751-754.
  7. Engler, C. et al. PLOS One. 2009, 4, 1-9.
  8. Anderson, J. et al. Sci Direct. 2006, 355, 619–627
  9. Arao, S. et al. Pancreas. 2000, 20, 619-627.
  10. Jiang, Y. et al. Sci Rep. 2015, 19, 1-9.


Venom, M.D.: How Some of the World’s Deadliest Toxins Fight Cancer


Venom, M.D.: How Some of the World’s Deadliest Toxins Fight Cancer

Nature, as mesmerizing as it can be, is undeniably hostile. There are endless hazards, both living and nonliving, scattered throughout all parts of the planet. At first glance, the world seems to be quite unwelcoming. Yet through science, humans find ways to survive nature and gain the ability to see its beauty. A fascinating way this is achieved involves taking one deadly element of nature and utilizing it to combat another. In labs and universities across the world today, scientists are fighting one of the world’s most devastating diseases, cancer, with a surprising weapon: animal toxins.

Various scientists around the globe are collecting venomous or poisonous animals and studying the biochemical weapons they synthesize. In their natural forms, these toxins could kill or cause devastating harm to the human body. However, by closely inspecting the chemical properties of these toxins, we have uncovered many potential ways they could help us understand, treat, and cure various diseases. These discoveries have shed a new light on many of the deadly animals we have here on Earth. Mankind may have gained new friends—ones that could be crucial to our survival against cancer and other illnesses.

Take the scorpion, for example. This arachnid exists in hundreds of forms across the globe. Although its stinger is primarily used for killing prey, it is often used for defense against other animals, including humans. Most cases of scorpion stings result in nothing more than pain, swelling, and numbness of the area. However, there are some species of scorpions that are capable of causing more severe symptoms, including death.1 One such species, Leiurus quinquestriatus (more commonly known as the “deathstalker scorpion”), is said to contain some of the most potent venoms on the planet.2 Yet despite its potency, deathstalker venom is a prime target for cancer research. One team of scientists from the University of Washington used the chlorotoxin in the venom to assist in gene therapy (the insertion of genes to fight disease) to combat glioma, a widespread and fatal brain cancer. Chlorotoxin has two important properties that make it effective against fighting glioma. First, it selectively binds to a surface protein found on many tumour cells. Second, chlorotoxin is able to inhibit the spread of tumours by disabling their metastatic ability. The scientists combined the toxin with nanoparticles in order to increase the effectiveness of gene therapy. 3 4

Other scientists found a different way to treat glioma using deathstalker venom. Researchers at the Transmolecular Corporation in Cambridge, Massachusetts produced an artificial version of the venom and attached it to a radioactive form of iodine, I-131. The resultant compound was able to find and kill glioma cells by releasing radiation, most of which was absorbed by the cancerous cells. 5 There are instances of other scorpion species aiding in cancer research as well, such as the Centruroides tecomanus scorpion in Mexico. This species’ toxin contains peptides that have the ability to specifically target lymphoma cells and kill them by damaging their ion channels. The selective nature of the peptides makes them especially useful as a cancer treatment as they leave healthy cells untouched.6

Scorpions have demonstrated tremendous medical potential, but they are far from the only animals that could contribute to the fight against cancer. Another animal that may help us overcome this disease is the wasp. To most people, wasps are nothing more than annoying pests that disturb our outdoor life. Wasps are known for their painful stings, which they use both for defense and for hunting. Yet science has shown that the venom of these insects may have medicinal properties. Researchers from the Institute for Biomedical Research in Barcelona investigated a peptide found in wasp venom for its ability to treat breast cancer. The peptide is able to kill cancer cells by puncturing the cell’s outer wall. In order to make this peptide useful in treatment, it must be able to target cancer cells specifically. Scientists overcame the specificity problem by conjugating the venom peptide with a targeting peptide specific to cancer cells.7 Similar techniques were used in Brazil while scientists of São Paulo State University studied the species Polybia paulista, another organism from the wasp family. This animal’s venom contains MP1, which also serves as a destructive agent of the cell’s plasma membrane. When a cell is healthy, certain components of the membrane should be on the inner side of the membrane, facing the interior of the cell. However, in a cancerous cell, these components, (namely, the phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) ) are on the outer side of the membrane. In a series of simulations, MP1 was observed to selectively and aggressively target membranes that had PS and PE on the outside of the cell. Evidently, using targeted administration of wasp toxins is a viable method to combat cancer.8

Amazingly enough, the list of cancer-fighting animals at our disposal does not end here. One of the most feared creatures on Earth, the snake, is also among the animals under scientific investigation for possible medical breakthroughs. One group of scientists discovered that a compound from the venom of the Southeast Asia pit viper (Calloselasma rhodastoma) binds to a platelet receptor protein called CLEC-2, causing clotting of the blood. A different molecule expressed by cancer cells, podoplanin, binds to CLEC-2 in a manner similar to the snake venom, also causing blood clotting. Why does this matter? In the case of cancer, tumors induce blood clots to protect themselves from the immune system, allowing them to grow freely. They also induce the formation of lymphatic vessels to assist their survival. The interaction between CLEC-2 and podoplanin is vital for for both the formation of these blood clots and lymphatic vessels, and is thus critical to the persistence of tumors. If a drug is developed to inhibit this interaction, it would be very effective in cancer treatment and prevention.9 Research surrounding the snake venom may help us develop such an inhibitor. .

Even though there may be deadly animals roaming the Earth, it is important to remember that they have done more for us than most people realize. So next time you see a scorpion crawling around or a wasp buzzing in the air, react with appreciation, rather than with fear. Looking at our world in this manner will make it seem like a much friendlier place to live.


  1. Mayo Clinic. (accessed Oct. 29, 2016).
  2. Lucian K. Ross. Leiurus quinquestriatus (Ehrenberg, 1828). The Scorpion Files, 2008. (accessed Nov. 3, 2016).
  3. Kievit F.M. et al. ACS Nano, 2010, 4, (8), 4587–4594.
  4. University of Washington. "Scorpion Venom With Nanoparticles Slows Spread Of Brain Cancer." ScienceDaily. ScienceDaily, 17 April 2009. <>.
  5. Health Physics Society. "Radioactive Scorpion Venom For Fighting Cancer." ScienceDaily. ScienceDaily, 27 June 2006. <>.
  6. Investigación y Desarrollo. "Scorpion venom is toxic to cancer cells." ScienceDaily. ScienceDaily, 27 May 2015. <>.
  7. Moreno M. et al. J Control Release, 2014, 182, 13-21.
  8. Leite N.B. et al. Biophysical Journal, 2015, 109, (5), 936-947.
  9. Suzuki-Inoue K. et al. Journal of Biological Chemistry, 2010, 285, 24494-24507.



Biological Bloodhounds: Sniffing Out Cancer


Biological Bloodhounds: Sniffing Out Cancer

50 years ago, doctors needed to see cancer to diagnose it - and by then, it was usually too late to do anything about it. Newer tests have made cancer detection easier and more precise, but preventable cases continue to slip through the cracks, often with fatal consequences. However, a new test has the potential to stop these types of missed diagnoses--it can detect cancer from a single drop of blood, and it may finally allow us to ensure patients receive care when they need it.

Blood platelets are a major component of blood, best known for their ability to stop bleeding by clotting injured blood vessels. However, blood platelets are far more versatile than previously understood. When cancer is formed in the human body, the tumors shed molecules such as proteins and RNA directly into the bloodstream. The blood platelets come in contact with these shed molecules and will absorb them. This results in an alteration of the blood platelets’ own RNA. Persons with cancer will therefore have blood platelets that contain information about the specific cancer present. These “educated” blood platelets are called tumor educated platelets, or TEPs. Recently, TEPs have been used to aid in the detection of specific cancers, and even to identify their locations.1

In a recent study, a group of scientists investigated how TEPs could be used to diagnose cancer. The scientists took blood platelets from healthy individuals and from those with either advanced or early stages of six different types of cancer and compared their blood platelet RNA. While doing so, the researchers found that those with cancer had different amounts of certain platelet RNA molecules. For example, the scientists discovered that the levels of dozens of specific non-protein coding RNAs were altered in patients who had TEPs. The further analysis of hundreds of different RNA levels, from the nearly 300 patients in the study, enabled the scientists to distinguish a cancer-associated RNA profile from a healthy one. Using these results, the team created an algorithm that could classify if someone did or did not have cancer with up to 96% accuracy.1

Not only could the TEPs distinguish between healthy individuals and those with a specific type of cancer, but they could also identify the location of the cancer. The patients in the study had one of six types of cancer: non-small-cell lung cancer, breast cancer, pancreatic cancer, colorectal cancer, glioblastoma, or hepatobiliary cancer. The scientists analyzed the specific TEPs associated with the specific types of cancer and created an algorithm to predict tumor locations. The TEP-trained algorithm correctly identified the location of these six types of cancer 71% of the time.1

The authors of the study noted that this is the first bloodborne factor that can diagnose cancer and pinpoint the location of primary tumors. It is possible that in the near future, TEP-based tests could lead to a new class of extremely accurate liquid biopsies. Nowadays, many cancer tests are costly, invasive, or painful. For example, lung cancer tests require an X-ray, sputum cytology examination, or tissue sample biopsy. X-rays and sputum cytology must be performed after symptoms present, and can often have misleading results. Biopsies are more accurate, but are also highly painful and relatively dangerous. TEP-based blood tests have the potential to both obviate the need for these techniques and provide more granular, clinically useful information. They can be performed before symptoms are shown, at low cost, and with minimal patient discomfort, making them an ideal choice to interdict a growing tumor early.

The information that TEPs have revealed has opened a gate to many potential breakthroughs in the detection of cancer. With high accuracy and an early detection time, cancer blood tests have the potential to save many lives in the future.


  1. Best, M. et al. Cancer Cell 2015 28, 676
  2. Marquedant, K. "Tumor RNA within Platelets May Help Diagnose and Classify Cancer, Identify Treatment Strategies." Massachusetts General Hospital. 


Microbes: Partners in Cancer Research


Microbes: Partners in Cancer Research

To millions around the world, the word ‘cancer’ evokes emotions of sorrow and fear. For decades, scientists around the world have been trying to combat this disease, but to no avail. Despite the best efforts of modern medicine, about 46% of patients diagnosed with cancer still pass away as a direct result of the disease.1 However, the research performed by Dr. Michael Gustin at Rice University may change the field of oncology forever.

Cancer is a complex and multifaceted disease that is currently not fully understood by medical doctors and scientists. Tumors vary considerably between different types of cancers and from patient to patient, further complicating the problem. Understanding how cancer develops and responds to stimuli is essential to producing a viable cure, or even an individualized treatment.

Dr. Gustin’s research delves into the heart of this problem. The complexity of the human body and its component cells are currently beyond the scope of any one unifying model. For this reason, starting basic research with human subjects would be detrimental. Researchers turn instead to simpler eukaryotes in order to understand the signal pathways involved in the cell cycle and how they respond to stress.2 Through years of hard work and research, Dr. Gustin’s studies have made huge contributions to the field of oncology.

Dr. Gustin studied a species of yeast, Saccharomyces cerevisiae, and its response to osmolarity. His research uncovered the high osmolarity glycerol (HOG) pathway and mitogen-activated protein kinase (MAPK) cascade, which work together to maintain cellular homeostasis. The HOG pathway is much like a “switchboard [that] control[s] cellular behavior and survival within a cell, which is regulated by the MAPK cascade through the sequential phosphorylation of a series of protein kinases that mediates the stress response.”3 These combined processes allow the cell to respond to extracellular stress by regulating gene expression, cell proliferation, and cell survival and apoptosis. To activate the transduction pathway, the sensor protein Sln1 recognizes a stressor and subsequently phosphorylates, or activates, a receiver protein that mediates the cellular response. This signal transduction pathway leads to the many responses that protect a cell against external stressors. These same protective processes, however, allow cancer cells to shield themselves from the body’s immune system, making them much more difficult to attack.

Dr. Gustin has used this new understanding of the HOG pathway to expand his research into similar pathways in other organisms. Fascinatingly, the expression of human orthologs of HOG1 proteins within yeast cells resulted in the same stimulation of the pathway despite the vast evolutionary differences between yeast and mammals. Beyond the evolutionary implications of this research, this illustrates that the “[HOG] pathway defines a central stress response signaling network for all eukaryotic organisms”.3 So much has already been learned through studies on Saccharomyces cerevisiae and yet researchers have recently discovered an even more representative organism. This fungus, Candida albicans, is the new model under study by Dr. Gustin and serves as the next step towards producing a working model of cancer and its responses to stressors. Its more complex responses to signalling make it a better working model than Saccharomyces cerevisiae.4 The research that has been conducted on Candida albicans has already contributed to the research community’s wealth of information, taking great strides towards eventual human applications in the field of medicine. For example, biological therapeutics designed to combating breast cancer cells have already been tested on both Candida albicans biofilms and breast cancer cells to great success.5

This research could eventually be applied towards improving current chemotherapy techniques for cancer treatment. Eventual applications of this research are heavily oriented towards fighting cancer through the use of chemotherapy techniques. Current chemotherapy techniques utilize cytotoxic chemicals that damage and kill cancerous cells, thereby controlling the size and spread of tumors. Many of these drugs can disrupt the cell cycle, preventing the cancerous cell from proliferating efficiently. Alternatively, a more aggressive treatment can induce apoptosis, programmed cell death, within the cancerous cell.6 For both methods, the chemotherapy targets the signal pathways that control the vital processes of the cancer cell. Dr. Gustin’s research plays a vital role in future chemotherapy technologies and the struggle against mutant cancer cells.

According to Dr. Gustin, current chemotherapy is only effective locally, and often fails to completely incapacitate cancer cells that are farther away from the site of drug administration where drug toxicity is highest. As a result, distant cancer cells are given the opportunity to develop cytoprotective mechanisms that increase their resistance to the drug.7 Currently, a major goal of Dr. Gustin’s research is to discover how and why certain cancer cells are more resistant to chemotherapy. The long-term goal is to understand the major pathways involved with cancer resistance to apoptosis, and to eventually produce a therapeutic product that can target the crucial pathways and inhibitors. With its specificity, this new drug would vastly increase treatment efficacy and provide humanity with a vital tool with which to combat cancer, saving countless lives in the future.


  1. American Cancer Society. (February 3 2017).                  
  2. Radmaneshfar, E.; Kaloriti, D.; Gustin, M.; Gow, N.; Brown, A.; Grebogi, C.; Thiel, M. Plos ONE, 2013, 8, e86067.                
  3. Brewster, J.; Gustin, M. Sci. Signal. 2014, 7, re7.
  4. Rocha, C.R.; Schröppel, K.; Harcus, D.; Marcil, A.; Dignard, D.; Taylor, B.N.; Thomas, D.Y.; Whiteway, M.; Leberer, E. Mol. Biol. Cell. 2001, 12, 3631-3643.
  5. Malaikozhundan, B.; Vaseeharan, B.; Vijayakumar, S.; Pandiselvi K.; Kalanjiam R.; Murugan K.; Benelli G. Microbial Pathogenesis 2017, 102, n.p. Manuscript in progress.
  6. Shapiro, G. and Harper, J.; J Clin Invest. 1999, 104, 1645–1653.
  7. Das, B.; Yeger, H.; Baruchel, H.; Freedman, M.H.; Koren, G.; Baruchel, S. Eur. J. Cancer. 2003, 39, 2556-2565.