Chip Synthesis

From Oligo Pool to Living Library

Chip synthesis results in a pool of single stranded oligos (A), designed with primer tags, which allow for the amplification of specific sub-pools, and Type II cut sites. qPCR, which amplifies the oligos at equal rates, results in the desired sub-pool (B). In order to prepare the oligo inserts and expression plasmid backbone for ligation, both must be cut with a Type II restriction enzyme (C, E). The cuts result in complementary sticky ends that anneal to each other during the ligation. The backbone, designed with a second cut site in the cut out gap (D), also undergoes a second digestion with a Type I restriction enzyme in order to minimize the number of undigested, or partially digested backbones. The oligo pool and backbone are then ligated (F), and transformed into library competent cells, resulting in a living library (G).

Zinc Finger Expression Plasmids

Expression Plasmid Schematic

The zinc finger expression plasmid was assembled through two-piece Gibson assembly, and contains the ColE1 origin of replication in addition to a spectinomycin resistance cassette. The zinc finger array is conjugated to the omega subunit of RNA polymerase, providing the basis for a one-hybrid system such that zinc finger target binding recruits RNA polymerase to transcribe downstream genes. In order to visualize and confirm zinc finger expression, GFP has been added to the end of the mRNA transcript, and is translated through the Shine-Dalgarno sequence AGGAGG that prefaces GFP by 7 base pairs. However, it must be noted that this only confirms expression of zinc finger mRNA without confirmation on the protein level. Zinc finger expression is controlled through the lac operator. Intrinsic activation of the lac operator has been found to be sufficient to induce zinc finger expression in significant amounts.

ZF Expression Plasmid

The expression plasmid contains a spectinomycin resistance cassette alongside a ColE1 origin of replication. Expression of the zinc finger array is controlled by a lac operator. The zinc finger array is conjugated to the omega subunit of RNA polymerase, which will serve as a recruiter for polymerase when the zinc finger array binds to its target DNA recognition site.

It should also be noted that GFP is used as a reporter for zinc finger expression, and its expression is controlled through the use of a Shine-Dalgarno sequence to cause GFP to be expressed as a separate protein from the same mRNA transcript as the zinc finger array.