Team:RAMNOTIREN CALGARY/Project/Content

PROJECT
• Overview
• Human Practices
• Safety
• References
• Attributions

Our Idea

Forty percent of Canadians are expected to develop cancer in their life time of that forty percent twenty five percent are expected to die from their cancer. Imagine what could be accomplished if all of those people were focusing their attention in their futures instead of the now? This question is exactly what motivated the Central Memorials IGEM team to create a bacterium that could target and kill cancer cells. We decided to combine the developing cancer treatment option of anti angiogenesis with synthetic biology to create a biological machine that would cut off the blood supply feeding cancer cells, effectively starving them. This method of killing cancer cells inspired us to title our project "Choking Cancer".

The Sequence

Our sequence consists of three parts: a lactic acid promoter, which is part BBa_K822000 on the IGEM registry, an anti-angiogenic factor, and a secretion tag. The lactic acid promoter activates the start codon, which promotes the production of angiostatin in the cell. A secretion tag will then be used to get the angiostatin out of the bacteria so that it can begin its inhibiting work on the blood vessels surrounding the tumor.

The Link between Cancer Cells and Lactic Acid

In the early stages of carcinogenesis, the tumor moves away from the blood supply and therefor its nutrient and oxygen supply. Many would assume that this would cause the tumor to experience hypoxia and nutrient shortages unfortunately this is not true. Cancer cells promote angiogenesis so that they can sustain their rapid proliferation and have a sufficient source of nutrients and oxygen. They also have a mutation that allows them to consume high amounts of glucose through a glycolysis pathway that, instead of sending glucose to the Krebs cycle, converts pyruvate to lactate. The large amounts of lactate secreted by tumor cells, as a direct result of the abnormal production of pyruvate, leads to the build up of lactic acid in the tumor surroundings. This build up of lactic acid is why our team chose to use a lactic acid promoter in our sequence. In doing so, our sequence will only become active in the presence of lactic acid and therefore near the tumor.

Why the Lactic Acid Promoter Was Chosen

The Central Memorial team chose to use bio brick BBa_K822000 (lactic acid promoter) because of the link between lactic acid and solid tumors described in the above paragraph. They wanted to choose a promoter that had been characterized and proven to be successful when used in sequences in they way BBa_K822000 has been. In doing this it allowed the team to focus less on how they were going to activate the start codon but instead on how they were going to develop the sequence, assays and protocols; as well as completing sufficient research on cancer and angiogenesis.

Angiogenesis

Angiogenesis is the process in which new blood vessels are formed from pre-existing blood vessels. Angiogenesis occurs in a healthy body during wound repair, to restore blood flow to tissues after injury. In women, it occurs during the monthly reproductive cycle as her body rebuilds the uterus lining, and during pregnancy, as angiogenesis helps to build the placenta and create blood circulation between mother and fetus. When the body is healthy, angiogenesis is managed by a series of “on” and “off” switches. The “on” switches are regulated by angiogenesis, stimulating growth factors such as Angiogenin, Angiopoietin-1, placental growth factor and vascular endothelia growth factor (VEGF) to name a few. The “off” switches regulated by angiogenic inhibitorssuch as Angiostatin (plasminogen fragment), platelet factor 4, thrombospondin -1 (TSP-1) and -2, and Troponin. If either Angiogenic stimulators of inhibitors are in excess, abnormal vascular growth occurs in the body.

The Link between Angiogenesis and Cancer

Cancer has a mutation that allows it produce small activator molecules of angiogenesis (research into what these activator molecules are is ongoing however it is thought that they are vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are both possible activator molecules). These activator molecules promote angiogenesis so that the cancer cell can continue to grow. If the cancer cell did not have increased blood flow to it, it would not be able to sustain its rapid proliferation. Research has been done that shows that without an increased blood supply, cancer cells cannot grow to more the 0.5 cubic millimeters. Therefore, if increased angiogenesis is the tipping point between a harmful cancer cell and a harmless cancer cell, it makes sense to tackle the problem using anti-angiogenic factors and synthetic biology.

Angiostatin - Our Anti-Angiogenic factor

Angiostatin is a cleaved portion of the protein plasminogen, and has been proven to inhibit angiogenesis. When angiostatin is cleaved from plasminogen, it is cleaved as a four-kringle domain. However, the most effective inhabitation of angiogenesis happens in only the first three Kringle domains. Our team’s sequence consists of angiostatin containing the first three-kringle domains. Angiostatin inhibits 38kDa, which specifically blocks the growth of endothelial cells. There was a slight modification made by ourteams in their cleaved portion of plasminogen. We changed amino acid 308N to 308E, which prevents glycosylation. In preventing glycosylation, a sugar chain is not added, and therefore the body does not recognize and destroy the protein immediately after it is secreted into the body. Once the angiostatin is secreted out of the bacteria into the body, it will cut off the blood supply feeding the tumor.

Secretion tag

The secretion tag will allow the angiostatin to be secreted out of the bacteria. It is important that angiostatin is secreted out of the bacterium so that the already heavy immune response to bacteria can be decreased. Secreting the angiostatin out of the bacteria allows for only the protein to leave the bacteria, while the waste and toxins remain inside. This is a better option than lysing the bacteria, which causes all of the bacteria’s waste to be released into the body along with the angiostatin. This would catalyze a sever immune response in the body, and possible sepsis. Please check back in 2015 to see how we will create the secretion tag.

Progress from January to June

Creating a bacteria sequence takes a lot of work and patience. A problem must be found and an idea developed. This idea would reveal how synthetic biology could be used to come up with a construct that would help bring the solution (to the problem) into closer reach. Central Memorial came up with the problem and theoretical solution by:

1. Finding a problem- this was cancer for our team
2. Doing some research into the problem- Which lead to the discovery of a developing treatment option that focused on angiogenesis
3. Researching this developing treatment option- in doing further research on anti angiogenesis Central Memorial found a way it could be combined with synthetic biology. They also found more information on cancer cells and how they are created. Including possible ways the cancerous tumor could be detected in the body by a bacterium.
4. Combing all the research- in doing so the team was able to come up with a sequence that consisted of a lactic acid promoter, which was chosen because in their research the team found that tumor cells secrete high amounts of lactic acid. When combing all the research they also came up with the idea to synthesize an anti-Angiogenic factor that would cause the blood vessels from reaching and feeding the tumor. The anti-Angiogenic factor they found was angiostatin. Their synthesized angiostatin contains 3 kringle domains cleaved from the original protein plasminogen. The original amino acid number 308N changed to 308E in order to prevent glycosylation and the protein being destroyed immediately after it was secreted.
5. Talking to the experts- Communicating with experts in synthetic biology, molecular biology and IGEM alumni allowed Central Memorials team to understand potential problems with their proposed idea, how these problems could be solved and future direction they should take. Potential problems, solutions and future direction are outlined below.
• Lysing the bacteria to get the angiostatin out would generate a strong immune response- The solution to this problem would be creating a secretion tag that would secrete only the angiostatin out.
• Muscles secrete lactic acid- the solution to this was for Central Memorials team to create a proof of concept idea. They would prove that bacteria with an anti Angiogenic factor such as angiostatin could be used to treat cancer cells. However in the future the sequence created could be inserted into a virus. This virus would contain a kill switch so that it would only be active and secreting angiostatin in the presence of a certain antibiotic. That way it could be used in conjunction with other treatment options.
• Bacteria in the body would create a sever immune response in an already sick body- A possible solution to this problem could be to insert the bacteria into the Nano tubes NASA is creating. These tubes will allow the angiostatin to be secreted out via the secretion tag, however the Nano capsule will contain the bacteria and keep the body from having an immune response to it.
• When the bacteria are injected into the body, how will it remain localized around the tumor? -This problem lead the team to an article explaining how a team of scientist at the University of California Berkeley, University of California and San Francisco have set out to create a tumor killing bacteria. This bacteria when injected into the bloodstream, would travel to the site of a tumor and insert itself into the cancer cell. Once inside the tumor it would produce a cancer- killing compound. In theory this cancer-killing compound could be our sequence. To read more about this bacteria click on this link.
6. Assays are designed and protocols carried out- Centrals team designed two assays that would be carried out the fallowing year to prove that their sequence would work. The first being one would show that the lactic acid promoter is active in the presence of lactic acid. Creating a circuit containing lactic acid and RFP would do this. To see exactly how this would be done proceed to the Assay section.
The second assay performed would show that the angiostatin is in fact an inhibitor of angiogenesis. Adding the bacteria to a kit plate that mimics angiogenesis would allow us to do this. The kit plate would change in some way to show that the inhabitation of angiogenesis took place when angiostatin was lysed from the bacteria onto the kit plate. To see exactly how Centrals team plans to carry this assay out proceed to the assay section.
The third and final assay will be performed to make sure that the angiostatin is produced in the presence of lactic acid. Adding the fully formed circuit to the kit plate that mimics angiogenesis with lactic acid will do this. The bacteria will cause the angiostatin gene to be turned on in the presence of lactic acid. If there is lactic acid on the kit plate then it will turn on the promoter causing the lactic acid promoter to turn on the angiostatin gene. When the angiostatin is lysed out of the bacteria it will cause the kit plate to change in some way are a result of its ability to inhibit angiogenesis. To see the specifics of this assay proceed to the assay section.
With the angiostatin in it to the In Vitro Angiogenesis assay kit we purchased will do this. This kit promotes angiogenesis or the formations of blood vessels. When this factor is active circles are formed on the kit plate, after adding angiostatin to the kit plates we hope the circles will disappear. If they do disappear then we will know that the angiostatin was successful. In order to ensure that it is in fact our protein causing the circles to disappear we will perform a few different tests involving the In Vitro Angiogenesis kit plate. First we will use the kit plate and add bacteria that do not contain our circuit. Hopefully the circles in the kit will stay intact, if they do we will know that it is not our bacteria causing the circles to disappear. Next we will add bacteria containing our angiostatin that have not been lysed. We hope that if all goes well then the circles will remain in the plate. This tells us that our factor is not somehow leaking out of the bacteria. Finally we will add our lysed bacteria to the kit plate (we will lyse the cells by adding detergent) and see if the circles disappear, if they do then our transformation of angiostatin into the bacteria was successful. The final essay performed will be performed in order to make sure that our circuit works correctly is quite simple. We will again use the kit plate we purchased that has an Angiogenic factor and put it into a well along with a concentration of lactic acid. This will be repeated five times with five different concentrations of lactic acid. If all goes well the circles should remain. In the next we will keep everything the same. However this time we will add bacteria. The next well will contain bacteria with our circuit inside however they have not yet been lysed and therefore the circuit will not be active. Again the kit plate and lactic acid is added to the well, and it is repeated five times. In the last well we will add our lysed bacteria and follow the same step we took for the previous sections. The well with the lowest concentration of lactic acid should have the greatest amount of circles however the amount of circles should be reduced compared to the wells where our circuit was not active. As the concentration of lactic acid increased the number of circles should decrease.
7. Carrying out protocols so that they will be done properly next year- This is done so that Centrals IGEM team can carry out all the procedures at the beginning of the year and get the circuit made by the end of December. They carried out a competent cell protocol, transformation method, growing a bacteria colony and feeding them, removing plasmids from bacteria and running a gel to verify plasmid size. To see these protocols in detail proceed to the protocol tab.

Cancer is a huge topic of conversation and with ongoing research progressing researchers knowledge of this disease and how scientist could put an end to it; Central Memorials team hoped that in completing the above steps they could spark a new topic of conversation. A conversation that one-day may lead to the end of this horrible disease.

Assays

In different concentrations of lactic acid different amounts of red fluorescent protein should be produced.
This kit promotes angiogenesis or the formation of blood vessels. When this factor is active. circles are formed on the kit plate; after adding angiostatin to the kit plates, we hope the circles will disappear. This would show us the success of the angiostatin. In order to ensure that it is in fact our protein causing the circles to disappear, we will perform a few different tests involving the In Vitro Angiogenesis kit plate. First we will use the kit plate and add bacteria that do not contain our circuit. Hopefully the circles in the kit will stay intact, if they do, it will confirm that it is not our bacteria causing the circles to disappear. Next we will add bacteria containing our angiostatin that have not been lysed. If the cirlces remain in the plate, it confirms that our factor isn't leaking out of the bacteria. Finally, we will add our lysed bacteria to the kit plate (we will lyse the cells by adding detergent) and see if the circles disappear, if they do then our transformation of angiostatin into the bacteria was successful.
The final essay performed will be performed in order to make sure that our circuit works correctly is quite simple. We will again use the kit plate we purchased that has an Angiogenic factor and put it into a well along with a concentration of lactic acid. This will be repeated five times with five different concentrations of lactic acid. If all goes well the circles should remain. In the next trial, everything will be kept the same; however, this time we will add bacteria. The next well will contain bacteria with our circuit inside. The circut, however,has not yet been lysed and therefore it will not be active. Again the kit plate and lactic acid is added to the well and it is repeated five times. In the last well, we will add our lysed bacteria and follow the same step we took for the previous sections. The well with the lowest concentration of lactic acid should have the greatest amount of circles however the amount of circles should be reduced compared to the wells where our circuit was not active. As the concentration of lactic acid increased the number of circles should decrease

What We Plan To Do

Our team plans to build three constructs. The first is a lactic acid promoter ligated with a red fluorescent protein. The second is a lactic acid promoter ligated with their angiostatin and the third is a lactic acid promoter ligated with angiostatin and a constituent promoter.

In the first assay a circuit containing a constituent promoter will cause the angiostatin to be continuously expressed in the bacteria. We will then lyse it and then run them in a western. On the western, a dot or a line should appear which shows that angiostatin is being expressed in the bacteria.

The second assay is performed to test the lactic acid promoter. A circuit comprised of the lactic acid promoter ligated with red fluorescent protein is inserted into the bacteria. These bacteria are then grown in different concentrations of lactic acid and then lysed. Next they are run on a western, the brightest line on the western shows at which concentration the lactic acid has maximum expression.

The third assay is executed to test the lactic acid promoter with the angiostatin. It is carried out to make sure that angiostatin is not toxic to the bacteria, or if it is toxic at what concentration is it toxic to the bacteria. The team also wants to see at what concentration of lactic acid could they get the maximum amount of angiostatin produced. To gather this information the team would put bacteria containing the ligated circuit containing a lactic acid promoter and angiostatin into different concentrations of lactic acid. The bacteria would be grown in these different concentrations of lactic acid and then lysed. Running a western would show Central Memorial at what concentration of lactic acid they got maximum induction of angiostatin (this would be the brightest line on the gel).

The fourth assay carried out is done to show the concentration of lactic acid needed to induce enough angiostatin to inhibit angiogenesis. This is done by growing bacteria containing ligated circuits of lactic acid promoter to angiostatin in different concentrations of lactic acid. The bacteria are then lysed and added to a kit plates containing vascular growth cells. The plate with tubes formed out of these vascular growth cells show angiogenesis and the plates without tubes formed shows that growth of the vascular growth cells was inhibited. The well with the fewest tubes formed would show the concentration of angiostatin needed to inhibit angiogenesis and at what concentration of lactic acid the concentration of angiostatin was produced.

The final assay performed would be carried to find a secretion tag capable of secreting the amount of angiostatin needed to inhibit angiogenesis. This would be done by putting a bacteria containing a circuit a ligated circuit of lactic acid promoter angiostatin and a secretion tag (several circuits would be created to put into bacteria containing different secretion tags) these bacteria would be grown in physiological concentrations of lactic acid (concentration of lactic acid produced by tumor cells), lysed and then run on a western. The secretion tag that secreted the concentration of angiostatin closest to that of the optimal amount needed to inhibit angiogenesis would choose. The optimal amount of angiostatin needed was determined in assay number four. The bacteria grown in the concentration of lactic acid producing optimal amounts of angiostatin (found in assay four) would be lysed on the far right corner of the western so that the secretion tag trials (which were run on the same western) could be compared to it.