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  2016 UIUC_Illinois iGEM Wiki

  
  
  
  
  
  
  
  

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Welcome to igem

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Description

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

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.