Chapter 15. Genetic engineering using SLIC - PowerPoint Presentation

1. Chapter 15. Genetic engineering using SLIC Sequence and ligase independent cloningYou can fuse any sequence to any other sequence(You could do this with BioBRICKS also and overcome the issue of the reading frame)

2. SLIC - sequence and ligase independent cloninghttp://j5.jbei.org/j5manual/pages/22.html+15 base 5’ additions to primers that are identicalto plasmid ends and PCRIn the absence of dNTPs,exonuclease activity of T4 DNA polymerasedigests 3’ ends (short incubation)addition of dCTP stops digestionat G in templateAfter annealing at room temp., the mixture is transformed into E. coli. single-stranded gaps are filled inand ends ligated by enzymes in E. coli.

3. How can we use DNA polymerases to make single stranded overhangs?You use polymerases with both polymerases and exonuclease activityYou can stimulate the endonuclease activity by adding the polymerase (plus buffer, water, and the vector or insert) but NO NUCLEOTIDEST4 DNA polymerase will generate a 5’ overhang with 3’-5’ exonuclease activityT5 DNA polymerase will generate a 3’ overhang with 5’- 3’ exonuclease activityThe exonuclease activity is timed to generate the desired length of overhang and then the reaction is stopped by adding a large amount of just one nucleotide.This stimulates the polymerase activityConsider why only ONE nucleotide is added. What would happen if we added a lot of all four nucleotides

4. 5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct tga3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga act 5’ E G R L Q K P L I E Y M V E R A - 5’ atg aca cgg cgg tgc tcg cacnnn3’ tac tgt gcc gcc acg agc gtgnnn M T R R C S H 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 SLIC to generate a gene that encodes an A+B fusion protein:1. to generate fusion ORF with these specifications: 2. ORF A 18 nucleotide (nt) right primer with 15 nt overlap, and 18 nt ORF B left primer without overlapORF AORF B

5. 5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct tga3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga act 5’ E G R L Q K P L I E Y M V E R A - 5’ atg aca cgg cgg tgc tcg cacnnn3’ tac tgt gcc gcc acg agc gtgnnn M T R R C S H Design primers to fuse by SLIC to generate a gene that encodes an A+B fusion protein:to generate fusion ORF – DELETE STOP CODON, MAINTAIN TRIPLET ORF FRAME 5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atg aca cgg cgg tgc tcg cac nnn3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc gtg nnn 5’ E G R L Q K P L I E Y M V E R A M T R R C S H ORF AORF B tga cac ggc ggt gct cgc acnnn act gtg ccg cca cga gcg tgnnnSTOPexample of consequenceof NOT maintaining ORF- deletion of A in ATG results in immediate STOP codonAs always, start with the end product you are trying to generate:

6. Design primers to fuse by SLIC to generate a gene that encode a A+B fusion protein:The fusion product you want to generate:ORF B5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atg aca cgg cgg tgc tcg cac nnnn3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc gtgnnn 5’ E G R L Q K P L I E Y M V E R A M T R R C S H This is the fusion site5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atg aca cgg cgg tgc tcg cac nnnn3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc gtgnnn 5’ E G R L Q K P L I E Y M V E R A M T R R C S H OverlapGene specific sequence

7. 5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct tga3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga act 5’ E G R L Q K P L I E Y M V E R A - 5’ atg aca cgg cgg tgc tcg cacnnn3’ tac tgt gcc gcc acg agc gagnnn M T R R C S H Design primers to fuse by SLIC to generate a gene that encode a A+B fusion protein:2. ORF A 18 nucleotide (nt) right primer with 15 nt overlap, and 18 nt ORF B left primer without overlap5’ 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 gtg 5’ E G R L Q K P L I E Y M V E R A M T R R C S H ORF AORF BORF A5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atg aca cgg cgg tgc tcg cac nnn3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc gtg nnn5’ E G R L Q K P L I E Y M V E R A M T R R C S H

8. 5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct tga3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga act 5’ E G R L Q K P L I E Y M V E R A - 5’ atg aca cgg cgg tgc tcg cac nnnn3’ tac tgt gcc gcc acg agc gag nnnn M T R R C S H Design primers to fuse by SLIC to generate a gene that encode a A+B fusion protein:2. ORF A 18 nucleotide (nt) right primer with 15 nt overlap, and 18 nt ORF B left primer without overlap5’ 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 ORF AORF BORF BORF A5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atg aca cgg cgg tgc tcg cacnnnn 3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc gagnnnn 5’ E G R L Q K P L I E Y M V E R A M T R R C S H After PCR (just the edges of the pieces that will be fused):5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atg aca cgg cgg tgc tcg 3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc5’ 5’ atg aca cgg cgg tgc tcg cacnnn 3’3’ tac tgt gcc gcc acg agc gtgnnn 5’

9. 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 ORF BORF AAfter PCR generating overlap:5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct atg aca cgg cgg tgc tcg 3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc5’ 5’ atg aca cgg cgg tgc tcg cac 3’3’ tac tgt gcc gcc acg agc gag 5’ After T4 DNA polymerase/exonuclease, exonuclease digestion: 5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atc gtc3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc 5’ 5’ atg aca cgg cgg tgc tcg cac nnnnnnnnnnnn3’ nnnnnnnn 5’After annealing of overlaps:5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atc gtc atg aca cgg cgg tgc tcg cac nnnnnnnnn 3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga tac tgt gcc gcc acg agc nnnnnn 5’ After transformation and repair in E. coli cells:

10. 5’ gag ggg agg ctg cag aaa cct ctc atc gaa tat atg gtc gag cgg gct tga3’3’ ctc ccc tcc gac gtc ttt gga gag tag ctt ata tac cag ctc gcc cga act 5’ E G R L Q K P L I E Y M V E R A - 5’ atg aca cgg cgg tgc tcg cac3’ tac tgt gcc gcc acg agc gag M T R R C S H Design primers to fuse by SLIC to generate a gene that encodes an A+B fusion protein:3. ORF B 18 nt left primer with 15 nt overlap, and ORF A 18 nt right primer without overlap 4. 18 nt primers with 15 nt overlaps generating a total of 30 nucleotides of overlap5’ 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 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 ORF AORF BDo these as homework – send me your answers and I’ll give feedback