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

Comments

Post a Comment

Popular posts from this blog

Mutagenesis Series: Overlap PCR

By TT -
Image
 Introduction PCR is a tool for used to multiply specific fragments of DNA, as an alternative to gene cloning,  that can be used to create gene variations in the laboratory. Overlap extension PCR is a technique that is commonly used for cloning large complex fragments, and fusing two gene fragments together.  In this series, Overlap PCR is used to fuse two gene fragments together.  As mentioned in previous postings.  The KanR gene from E. coli will be used to tag D. radiodurans and block the ZWF gene. The protocol used for this process uses touchdown PCR cycling and is completed in 2 steps. The first step was completed separately.  This posting will go over the 1 step of the Overlap procedure that was completed.  Procedure This protocol was optimized to eliminate the need for optimizing PCR cycling conditions.  There are no specific temperatures that must be used for the annealing cycle and the temperatures decrease on a gradient of 0.5 degrees Celsius per cycle.   This step was comp
Read more

Mutagenesis Series: 2-way Assembly

By TT -
Image
  Introduction Previously, step 1 of the Overlap PCR procedure was conducted. T here were no primers added to these reactions, which led to a gel that showed no bands of DNA because the fragments were not yet fused together.  They were only amplified.  The next step that will be discussed is called the Final Fusion step of this protocol.  This step should result in a 2-way assembly by fusing together the overhang sections created by the primers that will be added.  Procedure This protocol was optimized to eliminate the need for optimizing PCR cycling conditions.  There are no specific temperatures that must be used for the annealing cycle and the temperatures decrease on a gradient of 0.5 degrees Celsius per cycle.   This step was completed in two reactions.  This step required 22.5 uL of Q5 master mix, 4 uL of the step 1 reaction, 1 uL each of primers 1 and 4 (left fragment, tube1), 1u of primers 3 and 6 (right fragment, tube2), and 21.5 uL of PCR water.   These two reactions were put
Read more

Mutagenesis series: 3-way assembly pt 2

By TT -
Image
  Introduced  The first attempt at creating a linear strand was unsuccessful.  This gel looked like a previous gel from a prior stage of this experiment that had no primers added to the solution.  The assumption is that the primers were not added to the solution so another attempt was made and  primers 1 and 6 were  added to the solution. Procedure   Using the 2-way assembly of the right+middle, the addition of the left fragment was introduced to the solution that consisted of a 5X Q5 master mix, primers 1 and 6, and PCR water.  This process went through Overlap PCR and the Final Fusion steps of the Overlap PCR protocol.  The conditions of the thermocycler were set to the prescribed temperatures and durations that are detailed in the Overlap PCR protocol, which were the same as the step of creating the 2-way strand.  Results Gel of 3-way assembly attempt #2. Bands shown are about 2300 base pairs.   The attempt was successful.  The second attempt at creating a linear strand was successf
Read more