CDR3H tite-seq run
We obtained custom designed microarray oligonucleotides from LC Sciences to generate a library of mutant CDR1H (μ1H) and CDR3H (μ3H) domains. We created separate libraries for the CDR 1H and 3H regions by performing PCR on these oligonucleotide libraries. CDR3H mutants in the LC sciences microarray library were amplified by PCR using oRAL10 and oRAR10 primers to create μ3H sequences. CDR1 library sequences (μ1H) were created by amplifying the LC Sciences microarray library by PCR using oRAL11 and oRAR11 primers.   Non-mutated single CDR regions were created by performing PCR on the pJK36 plasmid with 1H 2F and 1H 1R primers to create the 1H primer, and 3H 2R and 3H 1F primers to create the 3H primer. By mixing non  mutated (1H or 3H) primers with library primers (μ1H or μ3H) and performing PCR on the pJK36 plasmid we created 360 bp long mutagenized scFv insertion sequences, iRA10, with one or both CDR regions mutated. The iRA10 sequence with added Bsal cut sites were made into iRA11 sequences libraries by performing PCR on oRA10 and oRA11 primers. The iRA11 sequences have complementary sticky DNA to the plasmid backbone pRA10 easing the integration of the sequence into the plasmid.   Having created library sequences, iRA11, that have sticky single stranded DNA at their ends after BsaI digest, we created a target plasmid for the iRA11 library sequences. The target plasmid pRA10 was designed to be have the same features as the pJK36 plasmid, but modified to have a deleterious ccdB gene that was toxic to DH10B E. coli growth, as well as the chloramphenicol marker, surrounded by BsmBI digest sites instead of the CDR1H to 3H domains of the scFv. The toxic ccdB gene served as a control, insuring that only E. coli that had pRA10 plasmids with iRA11 substitutions would grow. pRA10, was created from Gibson cloning iRA12, iRA13, and iRA14 sequences together. iRA12 contained the BsmBI digest sites, ccdB toxic gene, and chloramphinicol drug resistance markers. iRA12 was created by PCR from pJK14 (containing ccdB and chloramphinicol resistance) with oRA12 and oRA13. iRA13 was created by performing PCR on pJK36 with oRA15 and oRA16. iRA14 was created by performing PCR on pJK36 with oRA14 with oRA17. We transformed DB3.1 ccdB resistant E. coli with the pRA10 plasmid, grew them in Ampicillin and chloramphinicol, and then extracted the pRA10 plasmids from E. coli using the Qiagen mini-prep kit.   To introduce our mutagenized CDR1H and/or CDR3H sequences back into an scFv expressing plasmid, we substituted the ccdB gene in pRA10 with the iRA11 insertions containing our library mutations. To do this we, digested both the iRA11 library sequences and the pRA10 plasmid to create sticky DNA sequences that could be easily ligated together. We digested the pRA10 backbone with BsmBI and iRA11 with BsaI. Digesting pRA10 with BsmBI had the additional effect of excising the ccdB and chloramphenicol resistance genes from the plasmid. The BsmBI and BsaI digests were purified using PCR and mixed together at molar ratios of 1:2.5, respectively. The BsmBI and BsaI digest mixtures were ligated with T4 DNA ligase and then dialyzed to remove salts. Electrocompetant DH10B E. coli were electroporated with 50 μl of ligated DNA. E. coli were grown for 1 hour in SOB media, then Ampicillin was added and E. coli were grown overnight. Because ccdB is toxic to DH10B cells, only plasmid that were successfully ligated were able to grow. We used Qiagen’s mini-prep kit to extract amplified pRA11 libraries. We then chemically transformed EBY100 S. cerevisiae cells for each of the pRA11 libraries. Finally, we induced scFv expression in the transformed S. cerevisiae libraries. We FACS filtered cells which displayed both HA and Cmyc domains in roughly equal amounts to eliminate S. cerevisiae with indels in the scFv domain of their plasmids.