TITLE: Fabrication of DNA constructs by Gibson Assembly and Golden Gate reactions
AUTHORS: Tamara Matute,  Isaac Nuñez,  Peter Von Dassow,  Fernan Federici

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>We used this protocol to build a series of codon optimized BFP expressing vectors in the context of the EMS initiative from Gordon and Betty Moore foundation. A complete list of files corresponding to the DNA parts and vectors used in this study can be found at our </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Drive</span></a><span> and </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">zenodo</span></a><span> database.</span></div></div>

[STEPS]
?. [Introduction and rationale]
Golden Gate assembly creates vectors by combining vectors containing level 0 parts (e.g. promoters, CDSes, terminators) and an acceptor vector.  The first step involves the creation of libraries of level 0 parts by 'domesticating' DNA sequences of interest (e.g a promoter or a new fluorescent protein).
?. [Domestication of DNA sequences into level 0 part vectors]
Level 0 parts for Golden Gate reactions were generated by Gibson Assembly. Parts can be generated by PCRing natural sequences (e.g  genomic DNA) or existing vectors; or by fully synthesising (e.g. gblocks from IDT) DNA fragments of interest.For PCRed parts, reactions were performed with Phusion® High-Fidelity DNA Polymerase following this conditions: Thermocycling98°C  x 30 sec(98°C x 10 sec + 60°C x 30 sec + 72°C x 2 min) x 35 cycles72°C x 10 min4°C PCR mix:1ul primer forward Gibson1ul primer reverse Gibson1,33ul template (concentration: 1ng/ul)8 ul Buffer HF 5X0,8ul dNTPs (10uM)0,4ul Phusion® High-Fidelity DNA Polymerase (2U/ul)27,46 ul H20 Commericlaly synthetisised DNA fragments were ordered containing the 20-30bp overhangs used for Gibson assembly into the level 0 part vector.
?. [Gibson assembly step for the creation of level 0 part vector]
Desired PCR products were identified by gel electrophoresis and extracted with the purification kit Wizard SV Gel & PCR Clean-Up System (Promega). DNA concentration were corroborated with a Take3™ Micro-Volume Synergy™ HTX before performing Gibson reactions.The volumes used for each part were calculated by eq 1, 2 and 3.Eq.1Eq.2Volume ratio [ul] = pmol/required pmolwhere required pmol is:0,01 for parts >2500 bp0,03 for parts >200 bp and 0,05 for parts Volume to be added was calculated as:Eq.31,5µl of parts were combined with 4,5µl of Gibson Master Mix in ice. This reaction was incubated at 50ºC for 1 hour.Reagents: Gibson Mix (1.33X): 100 ul 5X Isothermal Buffer2 ul T5 Exonuclease (1 U/ul)6,25 ul Phusion® High-Fidelity DNA Polymerase (2U/ul)50 ul Taq DNA ligase (40U/ul)216,75 ul H20Store at -20°C.5X Isothermal Buffer:25% PEG-8000500 mM Tris-HCl pH 7,550 mM MgCl250 mM DTT1 mM dATP1 mM dTTP1 mM dCTP1 mM dGTP5 mM NADH20Store at -80°C
?. [Golden Gate assembly of promoters and fluorescent proteins combinations]
Final combinatorial assemblies of promoters, CDSes and terminators here described were produced by Golden Gate. For this, a general aceptor vector was created that accepts level 0 parts for the assembly of transcriptional units (e.g. promoter-CDS-terminator of interest).Eq.4The assembly was performed as follows:(37°C x 3 min + 16°C x 5 min) * 40 cycles.50°C x 5 min.80°C x 10 min4°C
?. [Transformation of Level 0 donor vectors]
These reactions were transformed into chemically competent TOP10 cells prepared by the OOW protocol (http://www.openwetware.org/wiki/TOP10_chemically_competent_cells), minipreped and sequenced. These level 0 parts were stored as vectors for the following Golden Gate reactions.
?. [Transformation of Golden Gate reactions]
3µl of these reactions were used to transform TOP10 Escherichia coli cells following the step described above.Positive colonies were identified by colony PCR and sequenced.