Team:BostonU/Mammalian synbio/Current Challenges

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Significance

Current Challenges

Proposed Solutions

Current Challenges

We aimed to identify a few broad reasons why mammalian cells are not heavily used in the iGEM community.

Cost

We very quickly recognized that there is a high cost associated with performing research in mammalian cells. This cost comes at many levels.

1. Higher Cost of Equipment and Facilities

   To use classic mammalian cell lines such as HEK293T cells, researchers require BSL2 certified spaces. The space itself may be difficult for individuals to obtain - working with multiple organisms in the same lab space may result in more chances for contamination, and thus for best results, researchers may want to have a designated space for mammalian use. Our iGEM team stems from a lab that has many researchers working with mammalian cells, and we were fortunate to be able to use dedicated BSL2 facilities for our research project.

   Some notable facilities and equipment needed for mammalian cell work include:

   1. CO₂ Incubator: In order to keep mammalian cells alive and growing, they need to be incubated at a certain temperature and CO₂ level. Our incubator was set at 37 C with a CO₂ level of 5.0%.
   2. O₂ Tissue Culture Hood: It is imperative to work in a sterile environment when working with mammalian cells to prevent contamination. The tissue culture hood provided this sterile environment for us to transfect, passage, and plate mammalian cells in. The tissue culture hood provides laminar flow of air to keep objects sterile, and also includes UV light sterilization after usage.
   3. Tissue Culture Plates: these are sterile plates upon which we plate mammalian cells for growth and transfection. The plates are treated with polystyrene such that cells can adhere on the bottom and spread out.
2. Higher Cost of Upkeep of Cells

   Mammalian cell lines require a lot of upkeep. To do transient transfection, cells need to be in an actively dividing state. However, if cells are left in the same tissue culture dish for a few days, they will revert into a quiescent state and will not be adequate for experimental purposes. Thus, researchers need to passage cells every 3-4 days, diluting them and transferring them into new dishes or flasks so that they are actively dividing and healthy.

   The process of cell passaging requires expensive reagents such as:

   1. FBS (fetal bovine serum) - promotes growth of mammalian cell culture
   2. DMEM (Dulbecco’s Modified Eagle Modicum) - media that supports growth of mammalian cell culture
   3. Penicillin Streptomycin (Pen-Strep) - helps prevent contamination in mammalian cell culture
   4. L-glutamine - amino acid that promotes cell growth in mammalian cell culture
   5. Sodium pyruvate - acts as an additional source of carbon and glucose for cell
   6. Trypsin - protease that cleaves mammalian cell surface peptides to release adherent cells from a tissue culture treated plate
   7. DPBS (Dulbecco’s Phosphate-Buffered Saline) - provides a buffer system to maintain media at optimal conditions

   We purchased most of these Corning products from Fisher Scientific.

Parts

Because sharing and submission of standardized parts to the common Registry is a core mission of iGEM, we sought to identify how mammalian part submissions might play a role in hindering teams from using mammalian cells.

1. Lower Number of Registry Parts

   We searched through the iGEM registry for functional parts that we could potentially use in our mammalian systems. iGEM teams generally tend to work in E. coli because it is easy to work with, grows quickly, and functions as a good host for foreign DNA sequences. Because of this, the vast majority of parts are designed to operate in E. coli. There is not a significant number of parts designed for optimal function in mammalian chassis. As a result, it is difficult for teams that are working with mammalian chassis to use and characterize old parts, as many of may not have full functionality in mammalian systems. This dissuades many teams from using mammalian chassis.

2. More Difficult Submission Standards

   There are two current avenues for teams performing mammalian research to submit their parts to the iGEM registry.

   The first is to submit parts as classic BioBricks. This ensures that parts are cloned and submitted in the pSB1C3 backbone for consistent iGEM handling, as well as that parts are flanked by proper restriction sites for subsequent cloning. One issue with this standard is that mammalian parts are quite large, and with many internal restriction sites it is difficult to remove these so that they are easily cloned as BioBricks.

   Our team noticed this problem during our own project. In order to clone some of our parts (~1-5kB) as BioBricks, we found many instances of 2-3 internal EcoRI, SpeI, XbaI, and PstI sites in these parts. We are thankful for the IDT offer that enabled us to eliminate some of these sites and synthesize our parts such that they were BioBrick compatible.

   The other option for part submission is to use the MammoBlock standard proposed by the MIT 2010 and 2011 teams. MammoBlocks utilizes a recombination-based assembly and is able to bypass the original BioBrick conformity requirements. However, cloning MammoBlock parts requires Gateway assembly, which is expensive and proprietary, and may therefore be infeasible for many teams.

Through our time working with mammalian cells we certainly came across different problems that hinder this research. Our iGEM team was fortunate enough to work in a lab that frequently utilizes mammalian cells. While we were extremely new to any procedures related to mammalian cells, we had many other researchers in lab who were there to help and guide us through the most optimal way to do things. We wish that everyone in the iGEM community had the same opportunities that we had, especially because so many teams do research for amazing health and medical applications. If we as a community were able to further that research into mammalian chassis, synthetic biology would be one step closer to feasible applications.

The biggest obstacle is definitely the cost associated with mammalian research. There is an initial cost to equipment and facilities and then an ongoing cost of upkeep of that equipment and the proliferation of cells. It is unfortunate that such a high cost is necessary for this cutting edge research, but we believe that as time goes on these costs will decrease because not only will new methodologies come out but even different techniques could arise. So, the most important thing we can do now is to begin the movement towards increased mammalian research by beginning a thorough discussion.

A concern more specific to iGEM is about the submission of mammalian constructs, which are generally larger and more internally complex than bacterial parts. If possible, we would want to lead further investigation into how to make BioBricks more welcoming for these parts or promoting use of MammoBlocks first suggested by MIT 2010 and 2011.

Finally, there is generally less familiarity and awareness within the community about using mammalian chassis for translational health applications. It is important to at least make teams aware about the pros and cons of this research so that they can actively decide whether or not their project would benefit from it.