Team:Paris Saclay/gibson


Gibson Assembly

Gibson assembly is a DNA assembly method which allows to join multiple DNA fragments together in a single, isothermal reaction. This method was invented by Daniel Gibson in 2009.

A single-tube Isothermal Assembly reaction features three different enzymatic activities that perform in the same buffer:

  • The exonuclease creates single-stranded 3´ overhangs that facilitate the annealing of fragments that share complementarity at one end (overlap region).
  • The proprietary DNA polymerase fills in gaps within each annealed fragment.
  • The DNA ligase seals nicks in the assembled DNA.

Isothermal Assembly works by combining a cocktail of exouclease, polymerase, an ligase to fuse dsDNA fragments with sufficiently (20-120 bp) homologous ends. It leaves no "scar" behind, i.e. you can expect your product to contain the EXACT over lap sequence. The reaction may work with shorter ends (e.g. 15 bp), so long as the annealing temperature is higher than 50°C.

To perform isothermal assembly:

1. PCR up your fragments of choice, and gel purify. Also gel purify the cut vector

2. Not exceeding a total volume of 5 µL, in a PCR tube, combine fragments at equal molecular ration [e.g. amount fragment 1 = 100 ng * (fragment size 1 / fragment size 2); amount fragment 2 = 100 ng* (fragment size 2 / fragment size 1)... etc.]. If required, bring to 5 µL with ddH2O. We recommend using approximately 100 ng of plasmid backbone.

3. Add the combined fragments (5 µL) to one Isothermal Assembly reaction aliquot (15 µL) and mix by pipetting (20 µL total).

4. Incubate samples in a thermocycler at 50°C for 60 minutes

5. (optional for chem. transformation)Purify with Qiagen PCR purification (MinElute) kit. Elute in 20 µL of ddH2O. [or simply dialyse]

6. The end result is a circular, double-stranded, fully-sealed DNA molecule that can be used to transform into Competent E. coli by using 10 µL of assembly mix

Making Isothermal Assembly Aliquots

5x isothermal assembly reaction buffer (assemble on ice):

From the paper: 	        Actually added: 
3 mL 1M Tris-HCl pH 7.5 	3 mL 1M Tris-HCl pH 7.5 
150 uL 2M MgCl2 	        300 uL 1M MgCl2 
60 uL 100 mM dGTP 	        600 uL 10 mM each dNTP 
60 uL 100 mM dCTP 	  
60 uL 100 mM dTTP 	  
60 uL 100 mM dATP 	  
300 uL 1M DTT 	                300 uL 1M DTT 
1.5 g PEG-8000                 1.5 g PEG-8000 
300 uL 100 mM NAD 	        20 mg NAD 
ddH2O to 6 mL 	                ddH2O to 6 mL 
  • Prepare 320 uL aliquots (18) and freeze all but one. * Label these “5X isotherm buffer”
To the one remaining (320 uL), add: 
1.2 uL 	T5 Exonuclease 
20 uL   Phusion polymerase (NOT HOTSTART) 
160 uL 	Taq ligase 
700 uL 	ddH2O 

Prepare 15 uL aliquots (~80) on ice in PCR tubes and store at -20C. These should be good for up to a year.

Shopping List Buffer

Item 	                                        Vendor 	                Cat. No. 
1 M Tris-HCl pH 7.5 	                        Systembio kitchen 	none 
Magnesium Chloride, 1.00 +/- 0.01M Solution 	Affymetrix / USB 	78641 10 x 1 ML 
Nicotinamide adenine dinucleotide (NAD) 	Applichem 	        A1124,0005 
DTT, molecular biology grade 	                FERMENTAS 	        R0861 
Polyethylene Glycol 8000, Powder 	        USB / Affymetrix 	19966 
dNTP Mix, 10mM each 	                        Fermentas        	R0192 1 ml 


T5 Exonuclease 	                        EPICENTRE   5E4111K 
Taq DNA Ligase 	                        NEB 	    M0208L 
Phusion™ High-Fidelity DNA Polymerase 	NEB 	    F-530S 

References: Gibson et al (2009) Nature Methods 6(5):343-345.