nanochromosome arrays combinatory assembly v1
Authors: Abramczyk Dariusz 
Published: July 2023

The yeast Pichia pastoris is used widely to biomanufacture high-value recombinant proteins. Its cells can secrete copious quantities of post-translationally modified proteins. Currently, however, heterologous genes must first be integrated into the genome in order to achieve expression. We produced a synthetic 'chromosome-like' construct called here nanochromosome, an autonomously replicating and mitotically stable synthetic construct expressing heterologous genes. The nanochromosome contains essential scaffolding features as telomeres, centromere and yeast replication origins along with a versatile platform for genetic engineering. This platform could be used for the accurate and controlled insertion of multiple expression cassettes placed on 'landing pad', in which an array of genes of interest alternate with ~1kp non-coding DNA sequences (LHR) chosen to facilitate simultaneous double cross-overhomologous recombination and serve as spacers. The landing zone translates along the nanochromosome in an inchworming mode of sequential gene integrations that recycles a pair of antibiotic-resistance markers Module with expression cassettes and LHR spacers as a pre-prepared in-vitro assembled DNA parts (insertion or integration arrays) is delivered to Pichia by transformation Both the “integration” (receptor) and “insertion” (donor) arrays referred to herein consist of long, HR-ready, regions (LHRs) alternating with gene-expression cassettes. We constructed array parts containing either two or three LHRs e.g.LHRn-GoIn-AMRH/Z-LHRZ or LHRn-GoIn-LHRm-AMRH/Z-LHRZ, respectively, wherein: LHRn, LHRm and LHRZ are unique sequences of ~1000 bp from a library of LHRsA-Z, with LHRZ reserved as the last LHR in the array; GoIn is a cassette consisting of a gene-of-interest with promoter and terminator regions; and AMRZ/H is a zeocin-resistance gene or hygromycin-resistance gene also with promoter and terminator regions. Preparation of ZeoR-LHRZ and HygR-LHRZ pair tandems in separate protocol https://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun The protocol for multi-DNA parts arrays preparation is based on combinatory assembly and molecular biology techniques customised to create a DNA parts (arrays) library in pUC19 plasmid. Major 3 experimental steps: Step 1 - DNA parts generated by PCR with a complementary overhangs designed to create a combined linear dsDNA suitable for insertion into BsmBI-sites of pUC19 (DNA arrays library) - a part generating by PCR, oligos deliver a complementary overhangs for digestion with BsmBI (Esp3I) or BsaI, - PCR product digestion with BsmBI or BsaI - checking digested PCR products on agarose gel - PCR clean up OR gel extraction of DNA and clean up (only in the case if a PCR product is not homogenous - estimation of DNA concentration (molar) Note: (*) stands for AsiSI-FspI RE recognition site provides in Fd-oligo1 and RV-olig4 Step 2 - preparation of semi-products before the the final ligation with linearlized pUC adaptor plasmid - ligation part1+2 with T7 DNA ligase - ligation part 3+4 with T7 DNA ligase - loading ligation mixtures and separation on agarose gel, - gel extraction of estimated DNA band - agarose gel verification, DNA concentration (molar) Step3 - the final ligation of pre-assembled parts with BsmBI-linearized pUC19 - part1+2 and part 3+4 and linearized pUC19 ligation (DNA T7 ligase) - transformation into E.coli and selection on LB ampicillin plates - verification by bacterial colony PCR - plasmid isolation and Sanger (Plasmidsaurus) sequencing - positively verified clone storage in glycerol stock The assembled part can be retained by: - PCR amplification using flanking oligonucleotides - Digested by AsiSI or FspI (need to be checked for the absence RE site in internal array) and digestion mixture can be used for Pichia transformation Note: LHR part (e.g. LHRA) in flanking region (as a part 1) sometimes called 'OUT' while the LHRA in internal site (as a part 3) sometimes called 'IN'