Golden Gate Assembly

GoldenGate frag1 frag2 frag3 BsaI product_name

Golden Gate Reactions

BsaI GGTCTC(N)₁ · CCAGAG(N)₅ — the cut lands one base past the site on the top strand, five on the bottom

5’-ATGCCATAGCATTTTTATCCATAAGATTAGCGGATCtGAGACCccatg-3’
3’-TACGGTATCGTAAAAATAGGTATTCTAATCGCCTAGaCTCTGGggtac-5’

                    5’-cactgGGTCTCGGATCTTACCTGACGCTTTTTATCGCAACTCTCTAC-3’
                    3’-gtgacCCAGAGCCTAGAATGGACTGCGAAAAATAGCGTTGAGAGATG-5’

↓BsaI cuts outside its own site, so both GGTCTC sites leave with the grey ends

5’-ATGCCATAGCATTTTTATCCATAAGATTAGCG-3’          5’-GATCtGAGACCccatg-3’
3’-TACGGTATCGTAAAAATAGGTATTCTAATCGCCTAG-5’          3’-aCTCTGGggtac-5’

5’-cactgGGTCTCG-3’              5’-GATCTTACCTGACGCTTTTTATCGCAACTCTCTAC-3’
3’-gtgacCCAGAGCCTAG-5’              3’-AATGGACTGCGAAAAATAGCGTTGAGAGATG-5’

↓T4 DNA Ligase — one GATC overhang on each piece, so they join one way only

5’-ATGCCATAGCATTTTTATCCATAAGATTAGCGGATCTTACCTGACGCTTTTTATCGCAACTCTCTAC-3’
3’-TACGGTATCGTAAAAATAGGTATTCTAATCGCCTAGAATGGACTGCGAAAAATAGCGTTGAGAGATG-5’

No BsaI site in the product — no scar. Then PCR with external oligos

EIPCR Mutagenesis

5’-…ATGCCATAGCATTTTTATCCATAAGATTAGCGAGACCccatg-3’
3’-…TACGGTATCGTAAAAATAGGTATTCTAATCGCTCTGGggtac-5’

               5’-cactgGGTCTCaTTAGTACcttACGCTTTTTATCGCAACTCTCTAC…-3’
               3’-gtgacCCAGAGtAATCATGgaaTGCGAAAAATAGCGTTGAGAGATG…-5’

EIPCR Mutagenesis

5’-…ATGCCATAGCATTTTTATCCATAAGATTAGTACcttACGCTTTTTATCGCAACTCTCTAC…-3’
3’-…TACGGTATCGTAAAAATAGGTATTCTAATCATGgaaTGCGAAAAATAGCGTTGAGAGATG…-5’

Design of a Golden Gate junction

Construct the sequences of the fragments you are joining
Pick the 4 bp junction — add 4 bases, take the last 4 of one fragment, or straddle the boundary
Copy and paste the BsaI sites and 5’ tails
Find ~20 bp of exact match past the junction — and past any mutation
Keep those two strands — and write the reverse one 5’ to 3’

5’-…ATGCCATAGCATTTTTATCCATAAGATTAGCGAGACCccatg-3’
3’-…TACGGTATCGTAAAAATAGGTATTCTAATCGCTCTGGggtac-5’

               5’-cactgGGTCTCaTTAGTACcttACGCTTTTTATCGCAACTCTCTAC…-3’
               3’-gtgacCCAGAGtAATCATGgaaTGCGAAAAATAGCGTTGAGAGATG…-5’

EIPCR

Design a Construction File to change the 5’ end of RFP in pBca9145-Bca1144 from atggcgagtagcgaagacg to atggctagtagcgaagacg without making additional mutations using BsaI EIPCR

Golden Gate from Plasmids

One vector per bin, each has matching, unique 4bp overhangs:

Nine small plasmids in a row, each carrying a spectinomycin marker and one part C1 to C9 flanked by a BsaI site, with a different four-base overhang written at each side: aggt, ccac, ggta, ccga, tacg, tgaa, atcg, atca, gaac, gctt. A tenth plasmid, pX-LacZ, carries a LacZ cassette between the same kind of sites. One arrow labelled one tube, BsaI plus ligase, leads to a single large plasmid, pGFPi, in which all nine parts sit in order between the same four-base junctions

Build basic parts in plasmids and sequence-confirm
Assembly them by pooling the plasmid DNA, then digest (BsaI) and ligate (T4 Ligase) simultaneously

Golden Gate Assembly

Golden Gate Reactions

EIPCR Mutagenesis

EIPCR Mutagenesis

Design of a Golden Gate junction

Golden Gate from Plasmids

Questions?