Team:Jefferson VA SciCOS/Results

Background

Wound healing is an integral part of everyday life. From a tiny paper cut to a large burn, wound healing is a vital life process that we depend on everyday. The process of wound healing is a process made up of four constructed steps. The first of these steps is called hematosis. During this stage of wound healing, vascular constriction occurs along with platelet aggregation, degranulation, and fibrin formation (thrombus). During the next step of the healing process, called the inflammatory stage, white blood cells eat away any dead tissue. This step also involves the formation of neutrophils, the infiltration of lymphocytes and monocytes, and the differentiation of monocytes to macrophages. The third step, proliferation, includes re-epithelialization, angiogenesis (the formation of blood vessels), collagen synthesis, and ECM formation. This step is very important in the wound healing process as it ensures that the wound will heal completely. The final step of wound healing is collagen remodeling and vascular maturation and regeneration.

Unfortunately there are several issues regarding wound healing that are not brought to public attention. Chronic wounds, wounds characterized by the fact that they do not heal within three months, present such issues as they do not heal in an orderly set of stages or a predictable amount of time. These wounds often tend to stay in the inflammatory stage for immeasurable periods of time. There are several factors that affect wound healing including but not limited to oxygenation, infection, invasion of foreign bodies, venous sufficiency, sex hormones, stress, etc. The data table below offers a more complete sample of factors that affect the wound healing process. There are many types of chronic wounds that have been identified over the years. However, two especially detrimental types of chronic wounds are venous ulcers and gangrene. Venous ulcers occur when the valves used in veins to prevent the backflow of blood, an integral part of the circulation of blood in the body, have been damaged, decreasing the pressure difference between the body’s arteries and veins. This condition is called venous hyperextension, and often causes the body’s veins to expand due to a large buildup of blood, pushing blood proteins into the extravascular space, which ultimately keeps growth factors from healing wounds that occur in the area. This can cause an extreme amount of pain in the affected area, which is most commonly located in legs. Venous ulcers are very costly to cure, which can be a major problem for low-income patients suffering from the chronic disease.

Gangrene is a condition in which blood cannot flow to an area of the body, resulting in a large amount of cell death. Gangrene can be caused by ischemia or by infection by certain bacteria including Clostridium perfringens. Often linked to long-term smoking and diabetes, gangrene often occurs in the extremities of the body, like toes. Treatment for gangrene is often difficult, as the most common treatments include surgery and amputation of the affected body part.

Testing

We hoped to determine KGF and FGF production by our transformed E. coli under hypoxic and anoxic conditions.

We will use the bacterial cell culture procedure on OpenWetWare to create our liquid cultures. We will use the spectrophotometer to measure our cell culture density at OD 600 and will use this information to adjust the cell culture density so that all of the tubes we use for the different trials are at the same density.

Oxygen Control System

To control the oxygen concentration of the liquid culture, we will use an oxygen controller to keep the oxygen level in the liquid medium at a certain level.  We plan to set the chamber at 0, 1, 2, 3, 4, and 5 percent dissolved oxygen for the different oxygen levels.  We will have multiple trials for each level.  After two hours at a given oxygen level, the cultures, which will be in tubes, will be centrifuged and frozen at -80 degrees Celsius to stop protein expression and cell growth.

Cell Lysis

The frozen cells will be resuspended in lysis buffer and the suspension will be incubated at room temperature for 20 minutes. The suspension will then be sonicated to shear DNA and reduce turbidity. The suspension will then be centrifuged at 40,000 x g for 20 minutes at 4 degrees C.

Protein quantification and measurements

We will quantify how much KGF is expressed under anoxic and hypoxic conditions using a human KGF ELISA kit, which uses a double antibody sandwich technique. Since growth factors are soluble, KGF and bFGF should be in the supernatant. We will pipette the supernatant out and into the wells of the ELISA plate. We will run the ELISA exactly according to the protocol given and will measure the amount of KGF captured and therefore expressed using a microplate reader at the given wavelength, which measures the color produced due to the enzymatic reaction.

Anticipated results - what we expect at different oxygen levels

We expected our bacteria to be able to respond to changes in oxygen levels by creating a maximum level of growth factor at a 2% oxygen threshold. When the oxygen levels are lower than 2%, we expected to observe a sharp decrease in production, and when oxygen levels are higher than 2%, we expected to observe a decrease in production in between the 2% and 3% threshold, with a linear increase in production for all thresholds above 3%. Oxygen thresholds below 2% should result in a sharp decrease in production of growth factor because the oxygen-dependent control mechanisms used by the promoter cause production to be maximally induced under hypoxic but not completely anaerobic conditions in E.coli. 2% is the ideal oxygen threshold according to the control mechanisms, but slightly higher thresholds should also produce significant yields.

Recommendations for further research and adjustments to protocols

To further study the relationship between oxygen levels and growth factor production using the constructed part, it is recommended that bacteria transformed with this part be tested at actual wound sites, so as to determine whether topical treatment using this application is feasible. In addition, more research regarding the chassis organism must be done before transformation in order to prevent an unnecessary immune system reaction against the membrane of the bacteria. Since the iGEM team was not able to apportion the time appropriately this year, it is recommended for future years that more planning and foresight be involved before experimentation. A good rule of thumb is that 90% of results in science are futile, so to maximize the chances of yielding satisfactory results, it is essential that everything is timed accordingly, everyone is aware of and experienced with protocols, and that no short-cuts are taken to obtain results. Also, the iGEM team plans to make trouble-shooting protocols for next year to use time more efficiently in case one of the steps is not performed correctly.

Future applications

We hope that our research will contribute to the development of “smart” wound dressings that accelerate healing and prevent infection. By combining synthetic biology with current research on the wound healing process and chronic wounds, we explored how bacteria could be used to produce growth factors in near-anoxic conditions. This result could be extremely useful for many facets of wound treatments. Of particular importance are chronic, hypoxic wounds such as gangrene. Research has shown that basic fibroblast growth factor (bFGF) has successfully treated gangrene in cases of both collagen disease and diabetes. A major cause of this success is bFGF’s promotion of angiogenesis in surrounding areas. Therefore, our bacteria could be useful in maximally producing angiogenesis-promoting growth factors in wounds that need it the most, i.e., in near-anoxic wounds. Our work could also be useful in acute wounds under near-anoxia, since it is important for growth factors to be produced early on in the healing process. In the future, these bacteria could be applied to many areas of medicine, ranging from acute wound healing to treatment of gangrene and other chronic, hypoxic wounds.

We were ultimately unsuccessful in our efforts to create a genetically engineered bacteria that would create growth factor in anaerobic conditions to aid in wound healing. However, our team gained powerful insight into our subject area. Our teacher and mentor, Dr. Burnett, once said that when you first start something, you should make as many mistakes as quickly as possible because that’s the best way to learn. We have certainly accomplished this and look forward to next year, when we can use what we learned to create a fantastic and impactful iGEM project!