"
Team:Wisconsin-Madison/
From 2013.igem.org
Cbjohnson4 (Talk | contribs) |
Cbjohnson4 (Talk | contribs) |
||
| Line 17: | Line 17: | ||
<div id = "divthecontent"> | <div id = "divthecontent"> | ||
<div id = "divtheoverview" style="height:auto;"><br> | <div id = "divtheoverview" style="height:auto;"><br> | ||
| + | <p align="left" class="classtheinlinecontent"><strong style="font-size:25px; color: rgb(183, 1, 1);">Expression and Purification of Enzymes</strong></p> | ||
<p class = "classtheoverview"> <strong>Gibson Assembly</strong></p> | <p class = "classtheoverview"> <strong>Gibson Assembly</strong></p> | ||
<p align="left" class = "classtheinlinecontent2">The Gibson Assembly utilizes three enzymes: (1) a thermostable DNA ligase; (2) a 5’-exonuclease; and, (3) a thermostable DNA polymerase, in an isothermal reaction to assemble together DNA fragments containing 20-40 base pair overlaps. The reaction proceeds as shown in figure 1: First, the exonuclease chews back the 5' ends of the overlapping pieces of DNA, allowing them to anneal. The amount of exonuclease must be finely tuned so that long enough sticky ends are created for the overlaps to anneal before the exonuclease becomes heat inactivated, but not a large enough amount of exonuclease that the DNA is destroyed. The polymerase then fills in the gaps created by the exonuclease until the ligase can join the strand, creating a single, unscarred, double-stranded piece.</p> | <p align="left" class = "classtheinlinecontent2">The Gibson Assembly utilizes three enzymes: (1) a thermostable DNA ligase; (2) a 5’-exonuclease; and, (3) a thermostable DNA polymerase, in an isothermal reaction to assemble together DNA fragments containing 20-40 base pair overlaps. The reaction proceeds as shown in figure 1: First, the exonuclease chews back the 5' ends of the overlapping pieces of DNA, allowing them to anneal. The amount of exonuclease must be finely tuned so that long enough sticky ends are created for the overlaps to anneal before the exonuclease becomes heat inactivated, but not a large enough amount of exonuclease that the DNA is destroyed. The polymerase then fills in the gaps created by the exonuclease until the ligase can join the strand, creating a single, unscarred, double-stranded piece.</p> | ||
Revision as of 19:59, 19 August 2013
Expression and Purification of Enzymes
Gibson Assembly
The Gibson Assembly utilizes three enzymes: (1) a thermostable DNA ligase; (2) a 5’-exonuclease; and, (3) a thermostable DNA polymerase, in an isothermal reaction to assemble together DNA fragments containing 20-40 base pair overlaps. The reaction proceeds as shown in figure 1: First, the exonuclease chews back the 5' ends of the overlapping pieces of DNA, allowing them to anneal. The amount of exonuclease must be finely tuned so that long enough sticky ends are created for the overlaps to anneal before the exonuclease becomes heat inactivated, but not a large enough amount of exonuclease that the DNA is destroyed. The polymerase then fills in the gaps created by the exonuclease until the ligase can join the strand, creating a single, unscarred, double-stranded piece.
Gibson, D.G. et al. Nat. Methods 343-345 (2009)
The Gibson Assembly is extremely useful for cloning. As the figure below shows, a gene can be designed to overlap a backbone plasmid. PCR can then be used to create the overlapping insert and backbone pieces, which can then be Gibson assembled.
Because of its simplicity and versatility, it is a very common cloning technique, however, the cost of the enzymes can be prohibitive for teams with small budgets. Therefore, we have provided an expression vector for T5 exonuclease and taq ligase, as well as a protocol for their expression and purification.
