A Look into Gibson Assembly

By TT -

What is Gibson Assembly?  

Gibson Assembly is a technique used to insert a single fragment or multiple fragments into a vector.  A vector or genetic vector is an instrument used as a delivery method to transmit foreign DNA to recipient cells.  The vector that will be used is the E. coli plasmid due to a particular interest in its kanamycin resistance gene.  Gibson Assembly allows gene transformation without the use of restriction enzymes.  Restriction enzymes are used as a defensive mechanism in bacteria against bacteriophages and can be used to manipulate genes within DNA.  


What are restriction enzymes? 

Restriction enzymes can be disruptive in gene manipulation.  These structures are meant to keep foreign entities out of the cell so it is possible that the restriction enzymes may cut in many sections of the gene causing a lack of overlap which would decrease the chances of ligation of the gene that is desired for insertion. 

What are the advantages and disadvantages of Gibson Assembly? 

Gibson assembly gives the executor control of the gene assembly and unwanted DNA sequencing can be avoided. 

A disadvantage of using Gibson Assembly is the ineffectiveness to assemble genes that are less than 100 base pairs and a decrease in efficiency when the number of inserts is increased.  

How will Gibson Assembly be used in this assay? 

Gibson Assembly will be used to insert the kanamycin gene from E. coli into Deinococcus radiodurans by knocking out the ZWF gene, or glucose-6-phosphate dehydrogenase, which is a protein that plays a major role in the pentose phosphate pathway in D. radiodurans. Once, the ligation of the kanamycin gene is complete, D. radiodurans response to oxidative stress will be evaluated.  


References 

SnapGene, May 4, 2021,  How to Simulate Gibson Assembly, https://youtu.be/uzaQr8QE0gY

Nature Portfolio, Genetic Vectors,  https://www.nature.com/subjects/genetic-vectors#:~:text=Genetic%20vectors%20are%20vehicles%20for,plasmids%2C%20viruses%20and%20artificial%20chromosomes.

Britannica, The Editors of Encyclopaedia. "nuclease". Encyclopedia Britannica, 3 Apr. 2014, https://www.britannica.com/science/nuclease. Accessed 2 April 2022.

Nova Online, April 2001,  https://www.pbs.org/wgbh/nova/genome/sequ_sans.html#:~:text=However%2C%20we%20don't%20want,there%20would%20be%20no%20overlap.&text=To%20limit%20the%20cutting%2C%20we,DNA%20at%20every%20possible%20site.

New England Biolabs Inc. 
https://www.neb.com/faqs/0001/01/01/what-are-the-advantages-of-this-method-compared-to-traditional-cloning-methods

Roth, T. L., Milenkovic, L., & Scott, M. P. (2014). A rapid and simple method for DNA engineering using cycled ligation assembly. PloS one, 9(9), e107329. https://doi.org/10.1371/journal.pone.0107329

Moors KA, Ott E, Weckwerth W, Milojevic T. Proteomic Response of Deinococcus radiodurans to Short-Term Real Microgravity during Parabolic Flight Reveals Altered Abundance of Proteins Involved in Stress Response and Cell Envelope Functions. Life. 2022; 12(1):23. https://doi.org/10.3390/life12010023

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