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Team:Lethbridge Canada/project
From 2013hs.igem.org
Contents:
Introduction:
Our project is to create a natural form of Oxytocin that can be used for widespread research and medical application. Oxytocin has a very short half-life, ranging anywhere from approximately three to ten minutes, in its active form (1). This means that over time, the hormone will degrade very quickly and become unusable. The goal is produce the hormone, attached to it's carrier molecule, Neurophysin I in order to prevent the breakdown of Oxytocin. We hope that our project will be able to make Oxytocin more readily available for study.
What is Oxytocin?:
Oxytocin is a hormone that has many effects on the body. Physically, it is known to stimulate uterine contractions, aiding the mother in birth (2). However, Oxytocin also has many other applications with social interaction. Oxytocin helps foster a bond between the mother and child, and stimulates a positive reaction when participating in social interaction. At present, Oxytocin is not comprehensively understood by researchers regarding its wide and varied effects. Many studies have been undertaken to determine exactly how Oxytocin interacts with the body. In some cases, Oxytocin provides results suggesting that it will enhance the social behaviours of animals and humans when added to their system (3). It is thought to improve facial recognition between face-to-face interactions, assisting in picking up on emotional cues(4). If true, Oxytocin could eventually aid people with social bonding disorders, such as autism(5), schizophrenia, and depression. Yet in some cases, it also produced results indicating individuals would isolate themselves into groups and promote exclusionary behavior(6). Researchers also do not have a strong case towards whether Oxytocin will have positive or negative effects in the human body when used long term, as so far all experiments have only dealt with short term effects on humans. All things considered, having cheap and efficient Oxytocin to study could greatly enhance our knowledge of the hormone, and eventually our ability to treat certain social disorders.
Parts:
Oxytocin-Neurophysin1:
We accomplish the task of synthesizing Oxytocin though the use of two separate constructs. The first, is a system designed from maximal gene expression in order to produce the greatest amount of hormone possible. In nature, Oxytocin is produced with its carrier molecule: Neurophysin I. This carrier protein inhibits the degradation of Oxytocin; prolonging its shelf-life. This combined compound is known as prepro-oxyphysin and is the gene of interest expressed in our system.
However, some modifications needed to be made to the gene during synthesis. First, a signal sequence native to E. coli, was added to allow for the protein to be exported beyond the cell due to the lack of Golgi Apparatus within the organism. The prepro-oxyphysin protein begins with a cysteine which forms a disulfide bond with with another cysteine later in the protein. In mammalian cells, prepro-oxyphysin is preceded by a signal sequence that guides it to the Golgi apparatus. This signal sequence is then cleaved from prepro-oxyphysin. We mimicked this by using the signal sequence PelB(8). This directed the prepro-oxyphysin through the inner membrane of E. coli and the signal sequence is cleaved as it passes through the membrane. This makes the first cysteine in prepro-oxyphysin available to for a disulfide bond. Additionally, histidine tags were added to the end of the protein to allow us to purify it using nickel-sepharose and detect it using mouse anti-his antibodies.
J23100_B0032_OXT_B0015
NEC1 Enzyme:
The function of the second construct is to produce NECI at a rate such that the numerical amount of NEC1 interacting with prepro-oxyphisin would be optimized. The aim of this to increase the efficiency of the system and to reduce the cost of producing NECI, as it is a very large protein and it may be difficult to produce in high amounts in E. coli. In order to do this, we made use of mathematical modelling to determine the correct ratio of enzyme to protein. Like the first construct, histidine tags were added after the enzyme in order to purify and detect it.
We are taking the first steps toward optimizing the expression rate of NECI by creating a model with different efficiency promoters followed by a high efficiency RBS and the fluorescent protein mCherry. We aim to monitor the expression rate of mCherry behind each promoter, and use this alonside our mathematical model to predict the amount of NECI we can produce. We will keep in mind that the NECI protein is significantly larger than mCherry when we are making our comparison.
Math Modeling:
For our project, we attempted to model the protein output of our cells. This would help us in finding the correct ratio of enzyme to protein to express. The full explanation as to how the math model works can be found on our Math Model page.
Visual Modeling
When I built the visual model, I encountered a few challenges. The first problem was converting the 3D PDB files using the software “VMD” to the proper format for Autodesk Maya (3D software) to work. Luckily, I was not the one who had to deal with the conversions because I wasn’t able to convert the file on my own anyway. Just downloading and converting the file took a whole week to figure out. I later found the plug in ePMV which was able to convert the files into Maya format. The second challenge was to learn enough of the Autodesk Maya I needed so that I could animate and pan around the proteins. My first tries were very jumpy and the animations were too dark. Finally I decided to place a camera on a motion track which was another too-complicated ordeal and with the camera on the track. After I added lights to either side, I had very smooth panning and bright conditions for the animation. My third problem was rendering the file. Before I did this project, I knew that rendering would be a pain, but I didn’t know how much. It took hours to set the render settings properly and hours after that to render the video to a 720p resolution. I was satisfied with my work however because it was smooth, not too fast, and looked professional enough in my eyes. My last problem was the worst problem. Frustrated doesn't even describe the video editing portion. In my mind I thought that what I rendered in the video was in the wrong order because it showed the NEC1 protein first and then the Oxytocin-NeurophysinI. So I reversed the video in Sony Vegas and added text, but I had issues with a lot of fragmenting in the video even after I rendered it. This resulted in approximately 5 hours of tweaking and trying to remove the massive amounts of fragments on the video. Finally, I scrapped the project and opened it up again to learn that it was because I reversed the video that it did that. After all of that I hammered out the video that is up on Youtube today and added some filtered music very quietly to have ambience to the video. I then rendered the video and placed it on Youtube.
- Joseph Adams
Conclusion:
In the end, we hope to produce a working Oxytocin-NeurophysinI (prepro-oxyphysin) construct along with a functional NEC1 construct. If we manage to attain those two constructs, we can obtain the hormone Oxytocin in its natural form. It is our hope that our construct will be able to substantially reduce the cost of producing Oxytocin in the commercial environment. Once we have constructed a working system, sending our parts into the parts registry will help future researchers study the effects that Oxytocin has in a mammalian system using the naturally occurring form of the hormone.
