Efficient Golden Gate Cloning Techniques
This content discusses the application of Golden Gate cloning method using BsaI restriction enzyme for seamless gene fusion. It provides detailed steps on primer design, PCR amplification, cleavage with BsaI, and ligation for efficient gene manipulation. The approach emphasizes working backwards from the desired gene product and designing primers with specific restriction sites to achieve accurate fusion. Understanding the unique features of Type IIS restriction enzymes is essential for successful implementation of this cloning strategy.
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Presentation Transcript
Golden gate cloning 1. Add to primers a. sequence identical to cloning site in plasmid (black) b. Bsa I site (red) 2. PCR amplify 3. Cleave amplicon and plasmid with BsaI 4. Ligate amplicon into plasmid no extra sequence added (seamless) fragment inserted in correct direction because sticky ends are different http://2013.igem.org/Team:Chiba/Parts
Type IIS Restriction Enzymes Differ from the type IIP REs we have been talking about These have sequences that are NOT palindromic These do not cut inside the recognition sequence, but to one side of it This means the overhangs generated will vary with each vector used You can design PCR primers with the restriction site on the 5 end and then add in the overhang sequence that will be generated after digest
Best Approach for the Design Start with the product you want to generate Then work backwards to see how you can generate it Finish with the design of the primers for your insert They will have the restriction site- we use BsaI most of the time- which will be cut off of the PCR product when we generate the overhang
BsaI Recognition sequence is GGTCTC (1,5) The (1,5) tells you where it cuts: 1 nucleotide 3 to the final C on one strand 5 nucleotides 5 to the first G on the other 5 -GGTCTCN/NNNNNNN-3 3 -CCAGAGNNNNN/NNN-5 5 -GGTCTCN NNNNNNN-3 3 -CCAGAGNNNNN NNN-5
Figure shows protein A end of ORF and beginning of protein B ORF (Figure ignores the rest of the genes and focuses on primers at fusion site) Design primers to fuse by Golden Gate method using BsaI to generate a gene that encodes an A+B fusion protein: ORF B ORF A 5 atg aca cgg cgg tgc tcg cac 3 tac tgt gcc gcc acg agc gag M T R R C S H 5 gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct 3 3 ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga 5 E G R L Q K P L I E Y M V E R A 1. to generate fusion ORF figure out what the product will look like: 5 gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atg aca cgg cgg tgc tcg cac 3 3 ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc gag 5 E G R L Q K P L I E Y M V E R A M T R R C S H 2. taking overlap sequence from ORF B
ORF B ORF A 5 atg aca cgg cgg tgc tcg cac 3 tac tgt gcc gcc acg agc gag M T R R C S H 5 gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct 3 3 ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga 5 E G R L Q K P L I E Y M V E R A 1. to generate fusion ORF figure out what the product will look like: 5 gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atg aca cgg cgg tgc tcg cac 3 3 ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc gag 5 E G R L Q K P L I E Y M V E R A M T R R C S H 2. taking overlap sequence from ORF B
Design primers to fuse by Golden Gate method using BsaI to generate a gene that encodes a A+B fusion protein: BsaI 5 -GGTCTCN 3 -CCAGAGNNNNN 2. taking overlap sequence from ORF B Take 4 nt sequence from end of ORF B, add to end of ORF A followed by BsaI sequence atg aca cgg cgg tgc tcg cac tac tgt gcc gcc acg agc gag M T R R C S H 5 ggtctcn 3 ccagagn 5 gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atgangagacc 3 3 ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tactnctctgg 5 E G R L Q K P L I E Y M V E R A - 3 -CCAGAGNNNNN 5 -GGTCTCN BsaI 5 gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atg aca cgg cgg tgc tcg cac 3 3 ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc gag 5 E G R L Q K P L I E Y M V E R A M T R R C S H
Design primers to fuse by Golden Gate method using BsaI to generate a gene that encodes an A+B fusion protein: 2. taking overlap sequence from ORF B After PCR : 5 ggtctcn 3 ccagagn atg aca cgg cgg tgc tcg cac tac tgt gcc gcc acg agc gag M T R R C S H After PCR : 5 gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atgangagagc 3 3 ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tactnctctgg 5 E G R L Q K P L I E Y M V E R A After BsaI digestion: 5 atg aca cgg cgg tgc tcg cac gt gcc gcc acg agc gag After BsaI digestion: 5 gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct 3 ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cgatact5 E G R L Q K P L I E Y M V E R A - After ligation: 5 gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atg aca cgg cgg tgc tcg cac 3 3 ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc gag 5 E G R L Q K P L I E Y M V E R A M T R R C S H
Design primers to fuse by Golden Gate method using BsaI to generate a gene that encodes an A+B fusion protein: 3. homework, repeat exercise, this time taking overlap sequence from ORF A ORF A 5 atg aca cgg cgg tgc tcg cac 3 tac tgt gcc gcc acg agc gag M T R R C S H 5 gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct 3 3 ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga 5 E G R L Q K P L I E Y M V E R A 5 gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atg aca cgg cgg tgc tcg cac 3 3 ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc gag 5 E G R L Q K P L I E Y M V E R A M T R R C S H
The fusion designs are repeated at the other ends of the PCR products to generate fusions with plasmid ends, or to generate multi-fragment fusions. There are many Golden Gate vectors available for many research organisms The CRISPR oligos you designed will eventually be cloned into a CRISPR vector designed for Golden Gate cloning
Example: Our CRISPR oligos will be annealed to form a double stranded molecule with 5 overhangs that match the BsaI cut cloning site in the vector
