Ethanol pool gRNA library at Ecoli stage, D3_Taq_20C_SR3
First, oligo pool consists of single stranded gRNA, BsaI restriction sites and common handle was synthesized from commercial company. A short oligo complementary the handle sequence was used to synthesize the second strand, with the PCR cycle being varied. These fragments were then digested with BsaI and ligated to vectors containing a promoter and terminator for gRNA expression. The resulting ligation products were introduced into E. coli DH5 cells, which were plated and cultured to form colonies. The plasmids were extracted from the agar plates. Direct plasmid sequencing was performed using SQK-RBK110.96.