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Team:RAMNOTIREN CALGARY/Notebook
From 2014hs.igem.org
- Week 18
- Week 17
- Week 16
- Week 15
- Week 14
- Week 13
- Week 12
- Week 11
- Week 10
- Week 9
- Week 8
- Week 7
- Week 6
- Week 5
- Week 4
- Week 3
- Week 2
- Week 1
Week 18
Today we effectively got the plasmids out of our bacteria by using our home made centrifuge. The white mass appeared at the bottom of our 1.5 ml tube. We centrifuged the plasmids by using a kit we purchased earlier this year. We followed the protocol given to us with the kit. When the white mass appeared a proud moment was shared between group members. We have overcome so many obstacles to get to this point.
Week 17
The Math department came through and managed to find us a drill that fit our 3D printer part. It also runs at a speed high enough to ensure the plasmid will be centrifuged out of the bacteria. This is exciting and means that we can finally get the next stage of our project going! At todays meeting we split into two teams. One team set up the centrifuge and the other team started to dissect the protocols. We hoped that by dissecting the protocols and going through them with a fine toothcomb they would be able to make more sense of them. Lucky for us both teams were successful and we finally have an idea on how we are going to carry out the next part of our project.
Week 16
We have run into a few problems and as a result our project is at a standstill:
The first of these problems being we don’t have a centrifuge and therefore we can't get the plasmids out of our bacteria, which is the next step in our process. Currently we are trying to locate a 3D printer piece that will fit on a dremmel drill. The 3D printer piece will allow us to attach 1.5 ml tubes to the dremmel drill. When the drill is running it can get up to 10000 RPM, as speed fast enough for us to centrifuge our bacteria with. However we can't find a 3D printer piece that will fit onto the drill we have. Therefore we have recruited one of the math teachers at our school to see if he has a drill that will fit our piece.
The second problem is our group is having a very hard time comprehending the protocols. Our group is comprised of only relatively smart students with a passion for biology. Notice I say passion for biology not a PHD in biology. The reason we are having so much trouble understanding the protocols is because we have lost our leading teacher for the time being. However the team is determined to get through this situation. In fact we have planned a meeting that will just be focused on reading the protocols dissecting them. Hopefully after we do this we will be able to figure out what we need to do next and how it’s going to get done.
We are also running out of pipette tips and therefore we have to be really stingy with them. When we re-home our bacteria, we have to make sure the tip don’t touch anything but the LB broth when we are filling our nine 1.5 ml tubes with it. This tip is then discarded and a new one is used for the chloramphenicol making sure that the new tip doesn’t touch anything but the solution as we fill the nine 1.5 ml tubes. It is only when transferring the bacteria from one tube to the other do we change tips. This is because we don’t want to cross contaminate the bacteria and get different types growing in the same tube. Currently we are trying to find a teacher who has the time to take some more pipette tips down to the u of c to get autoclaved, however this is no easy task. A student can’t do it as the CBE (Calgary Board of Education) won't allow it.
Week 15
We have returned to the books and are frantically trying to get everything in order for the conference at the U of C; we created a presentation. The reason this is so important is because the conference is going to help better prepare us for the competition in Boston. We need to figure out where our research is lacking and if the presentation conveys our idea and information to an audience well. We are also working on cementing the assays we intend to carry out this year and into the beginning of the next school year. It was a fast paced meeting and we didn’t leave a lot of room for chatting and laughs. We went to improvement meetings and talked about our ideas. We are grateful for today.
Week 14
We just started re-homing the lactic acid promoter, red fluorescent protein, and competent cells. They take over your life and currently all they do is eat and produce waste. Or at least that’s all they seem to do. Either way it is exhausting to move them every day. However , It is amazing to have begun the microbiology processes. Soon, we will be centrifuging the plasmids out of the bacteria so that we can cut and ligate them. This process usually takes about 10 minutes but after many trials, it takes about 30-45 minutes to re-home all the specimen.
Our leading teacher just had a baby and we are very happy for his family.
Week 13
We finally got started on some of the actual molecular biology! Although it was pretty basic stuff (or at least basic to those who have made a career out of this) it was super cool. We rehydrated the lactic acid promoter and the red florescent protein and using the heat shock method transferred the proteins into the competent cells that David Lloyd gave to us. For those of you reading this and don’t speak the molecular biology language what I said above means that we got our bacteria to express traits that are not genetic to the strain. However we added proteins to the bacteria so that they could express the traits we desired but we managed to get it finished.
Week 12
Today we meet with our mentor David Lloyd; it was a great and productive. David helped us figure out the assays we are going to perform with the bacteria we have made. We learned that we would need to run two very important essays in order to verify a proof of concept with our bacteria. We will need to put the bacteria containing our ligated circuit of the lactic acid promoter and red fluorescent protein and into different concentrations of lactic acid. In these different concentrations we will see different amounts of red fluorescent protein expressed. In seeing this we will prove that our circuit works. Once we have proven this circuit to be accurate we can then ligate our lactic acid promoter to the angiostatin protein. We will test this circuit by putting it into the kit plate containing green circles and different concentrations of lactic acid. The plate with the highest concentration of lactic acid should contain the least amount of green circles, as the highest amount of angiostatin should have been released. The green circles disappear in the presence of angiostatin.
We also began to discuss the presentation how to get ready for both the u of c and the competition in Boston. David talked to us about how we should engage the audience as well as making a presentation that flows smoothly and is easy to follow. He especially emphasized that we need to make sure to connect everything in the presentation back to our main idea and goal. David also told us to make sure that we don’t throw the less scientific things into our presentation at the very end. This would look rushed, messy and de value all of the hard work we have done of things like human practices. Overall this was an extremely useful meeting and we are now finally ready to start on some of the actual molecular biology.
Week 11
Today was a very slow day as we had a low turnout and so, we got to walk the runway and pretend we were bio superstars! At our meeting we did a photo shoot so that we could add some fun photos to the web page. It was a blast; we got to wear the purple lab coats all the teachers at our school use and all of us were looking pretty snazzy. It also got me really excited to actually start creating our bacterium!
Week 10
At this meeting we went through some papers which helped us find a section of cleaved Angiostatin which would be between the kringle domains 1-3 because of its anti-inflammatory factors. We talked about protein domains. There have been trials that have shown that this mutation within the DNA can prevent glycosylation--> adding a sugar chain to protein which targets it for degradation, so breaking up and getting rid of the bacteria. We talked about negatives of Angiostatin and positives. The exciting part is that we have the version of the section of Angiostatin protein that we want, we fixed and re wrote it to have the mutations we want, and talked about recombinant DNA and how we can cut the codes that we want and put it into our vector to create our bacteria.
Week 9
We spent the meeting sifting through research on Angiostatin- specifically how it is cleaved, its shape and its DNA sequence- with Mr. Garg. It was a HUGE help having Mr. Garg as he was able to explain some of the more technical parts of the papers we were looking at.
In one of our first meetings, we established committees to try and get the project moving forward in a more efficient manner. Our business committee were tasked with try to get companies to sponsor us. We have to start contacting sponsors as soon as possible. Everyone is worried that we have left it to the last minute. Over the past few weeks, we have been searching for companies that would be interested in sponsoring us and compiling a list of names, addresses and phone numbers we could send out or sponsor letters too. At today’s meeting, we began to call companies so we could try and find out whom sponsor letters should be addressed to. Everyone we spoke to was very friendly and we only got hung up on once.
Week 8
Upon our research, we came up with three proteins that we could get bacterium to secrete to promote anti angiogenesis. The most promising one was Angiostatin as opposed to Platelet factor four which is essentially just a blood clotting protein and Thrombospondin, which is very complicated and has been a challenge for everyone who has tried to research it. Consequently, we have decided to go ahead and focus our attention solely on Angiostatin for now. At the end of today's meeting, we decided to begin to research Angiostatin further; specifically if the protein could be secreted. This is crucial because if it cannot be secreted then we cannot use it in our bacteria.
Week 7
At today's meeting, we worked on our abstract. We also went over some papers that brought up the idea of TNF, IL-12 and Avastin as possible proteins but we had to look further into them to find out if they were a protein or synthetically made. We also discussed where it would be better to work out bacteria towards retracting cancer or stopping it and what were the pros and cons.
Week 6
We discussed more about what protein and promoter we wanted to use, as well as how to transport the bacteria into the cancer cell. Some question that needed to be answered was what are the downfalls of the bacteria going everywhere in the body? And are there any vaccines, cure or drugs that assist angiogenesis? We talked about Angiostatin and its positives and negatives. The positives were that it is already used as a cancer therapy, has no resistance, and can be used in plasma. The negatives are you can’t use it if you have high blood pressure and whether or not it could be cleaved. We had to look into the cleavage of Angiostatin and whether or not it can be activated when secreted into the blood and if it should be cleaved before or after. We discussed if it will clot in the blood, the length of the amino acids if it were to be pre cleavage, and more about the side effects.
We also talked about the human practice aspect and the online survey we had set up and its results. Overall we decided we needed to look more into the public’s openness for treatment, their openness for funding and the concern about physical activity and what the rate of physical activity would be. - Traelyn
Week 5
It is time to get the human practice section underway! At today's meeting we created a list of eight survey questions that we are going to take to the public. These survey questions are supposed to help us determine how accepting the public will be of our idea. We are trying to figure out if someday, given the option, individuals would choose to have bacteria inserted into their blood stream instead of undergoing chemotherapy to fight cancer.
Here is a list of the survey questions:
1.Have you or someone you know ever been affected by cancer?
2.Would you be comfortable having bacteria injected into your blood if it could treat cancer?
3.Would you support your taxes going to this cause?
4.Would you be okay with side effects such as fatigue/flu-like symptoms?
5.Would you be okay with this form of treatment if the duration was the same as traditional treatment options?
6.If this form of treatment took twice as long but had a higher success rate, would you still opt for this form of treatment?
7.Assuming similar success rates, would you prefer chemotherapy or would you use the bacteria?
8.During treatment, would you be okay with no physical activity?
We also discussed how we should explain the project to other people so that they understand what we are trying to do. This was not an easy task. To make matters even more difficult, we would have a very limited amount of time to explain our project. Near the end of the meeting, a couple of the other group members managed to find a succinct way to explain the project. Now the only thing that is left is to try and figure out how we are going to bring our survey to a large population of people. - Maggie
Week 4
Now that we have officially decided that we want our bacterium to fight cancer by promoting anti angiogenesis, it is time to focus our attention on finding a protein that will do this. What will happen is this: once we find the protein that can promote anti angiogenesis, we will insert it into the bacteria and get the bacteria to secrete the protein in the presence of lactic acid. Now is that cool or what? We are currently trying to gather a list of all the possible proteins that could promote anti angiogenesis, and while this is a tedious task, we are getting through it. Once the list is put together, we have to go though it and try to weed out the most promising proteins.
[EDIT] We talked about what our genetic circuit would be, discussing what bio bricks were and how we would need a promoter and a gene bio brick. These would code for out protein which at this point we had not decided what anti-angiogenesis protein we would use. We discussed the idea of using target therapy. - Traelyn
Week 3
Now that we have decided on the type of biological machine we want to create, it is time to start doing some research. The group talked and we have decided we would like our bacterium to choke (for lack of a better word) cancer cells. We would do this by creating bacteria that contains an anti angiogenesis inhibitor, thus causing the blood vessels to retract and the cancerous tumour to starve.
We also watched a TED Talk (http://www.youtube.com/watch?v=C_5Z31mUmtc) During this meeting that better explained anti angiogenesis and the success different scientists have had in treating cancer with anti angiogenesis drugs. It is truly amazing what some have been able to do! For example, there was this one experiment done on a dog with a Malignant Neurofibroma tumour growing on its shoulder that had spread to its lungs. Milo the dog had only a few months to live, however after being given a cocktail of anti antigenic drugs, the growth of the tumour slowed. In the end, the scientists were able to extend Milo's life by six times what the veterinarian had predicted. Now that is incredible!
After finishing the TED talk, we ended the meeting. Everyone was supposed to go home and do some more research on anti angiogenesis. - Traelyn
Week 2
After some discussion, the majority of the group decided we needed to come up with some different project ideas. It turns out that our seemingly brilliant idea to create a biological machine that can help celiacs digest gluten wasn't so brilliant. An IGEM team in 2011 engineered an organism to do just that, making it a completely unoriginal idea (and we want our idea to be original). So sadly, we had to go back to the drawing board and start thinking of some new ideas. Finally, after some very roundabout conversations, it hit us. We wanted to create a bacterium that could detect and help fight cancer! - Jamie
Week 1
Today was my first IGEM meeting and man oh man was it exciting! Synthetic biology is probably one of the coolest things I have ever heard of, and I can't believe I'm getting the chance to divulge deeper into this incredible science. I mean, looking at what groups are trying to accomplish with synthetic biology is mind blowing. I especially liked the bacteria Lethbridge's IGEM team created; what a great idea to try and get a bacterium to produce insulin for people who can't. I hope that our team can come up with an idea for a bacterium that could potentially help people that are suffering from medical conditions. So far we have discussed looking into creating either a biological machine that could break down gluten or an exothermic bacteria that could help melt ice. - Jamie
