Team:UIUC Illinois

The development of molecular cloning techniques was a turning point in the fields of molecular biology and genetics. It suddenly allowed scientists to isolate and study individual genes from a larger system. Molecular cloning is the process by which recombinant DNA molecules are made and transformed into a host cell, where they are then replicated. Two components are necessary for a molecular cloning reaction to occur.

  • A DNA segment of interest to be replicated
  • A vector/plasmid backbone that contains all of the elements needed for replication/expression in the host


Traditional cloning protocols used restriction enzymes to excise the DNA of interest from its source DNA. The DNA fragment was then amplified and joined to a plasmid backbone. The plasmid is a small, circular piece of DNA that is replicated within the host, and exists separately from the host’s chromosomal or genomic DNA. Physically joining the DNA of interest to the plasmid vector ensures that the DNA of interest will be fully integrated in the recombinant plasmid and thus replicated by the host.


Although traditional cloning methods were revolutionary, there were several caveats to the process that set the stage for an updated, more efficient version of this technique: Gibson Assembly. Gibson assembly is a method of cloning that capitalizes on the properties of 3 common microbial enzymes; 5' exonuclease, polymerase and ligase.

  • 5' exonuclease digests the 5' end of double stranded DNA to generate 3' single-stranded overhangs. The newly generated ends feature an area of 20-40 base pairs that is homologous to two or more DNA fragments in the target plasmid. Thus, the exonuclease creates complementary "sticky ends" which will efficiently find the homologous DNA pieces and anneal. The sticky ends are similar to the ends generated when using restriction enzymes except that these sticky ends have a larger region of complementarity.
  • DNA polymerase fills any remaining sections of single-stranded DNA after the DNA sections have annealed.
  • DNA Ligase then joins the segments into one continuous DNA fragment by filling in any gaps or nicks.


Unlike traditional restriction enzyme methods, Gibson Assembly allows one to simply assemble multiple fragments of DNA in any chosen orientation without any unwanted or unnecessary DNA fragments at the junctions. Though Gibson Assembly offers a more efficient and effective cloning process, its high cost makes it pragmatically difficult to use.

 

Unfortunately, the reagents necessary for Gibson are costly and often out-of-budget for many labs who are instead forced to use less successful and more time-consuming traditional approaches. If Gibson Assembly were more widely accessible, the impact of the scientific community as a whole would be profound. Accessibility could facilitate a higher level of discovery, understanding and progress. Through the implementation of synthetic biology principles and techniques, we aim to eradicate this barrier to scientific progress by engineering a cheaper, more efficient form of Gibson Assembly.

Wiki template information

We have created these wiki template pages to help you get started and to help you think about how your team will be evaluated. You can find a list of all the pages tied to awards here at the Pages for awards link. You must edit these pages to be evaluated for medals and awards, but ultimately the design, layout, style and all other elements of your team wiki is up to you!

Tips

This wiki will be your team’s first interaction with the rest of the world, so here are a few tips to help you get started:

  • State your accomplishments! Tell people what you have achieved from the start.
  • Be clear about what you are doing and how you plan to do this.
  • You have a global audience! Consider the different backgrounds that your users come from.
  • Make sure information is easy to find; nothing should be more than 3 clicks away.
  • Avoid using very small fonts and low contrast colors; information should be easy to read.
  • Start documenting your project as early as possible; don’t leave anything to the last minute before the Wiki Freeze. For a complete list of deadlines visit the iGEM 2017 calendar
  • Have lots of fun!
Inspiration

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