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This protocol describes the materials and method to conduct an experiment of Variant Abundance by Massively Parallel sequencing (VAMP-seq) assay. There are two designation plasmids available for a gene of interest (GOI) to fuse with a EGFP reporter system fused either N-terminally (N-terminal; EGFP-GOI) or C-terminally (C-terminal; GOI-EGFP).
Guidelines
Pre-barcoded Vector Design:
The current (as of 2023) working system (VAMP-seq v2) is modified based on the published version (Matreyek et al, 2018). There are two major modifications made for a two-step “plug and play” system compatible with Golden Gate Assembly. First, a pair of BsmBi/ Esp3I recognition sequences were added before the T7 promoter sequence in both of the N- & C- terminus systems. This is used to add barcode sequences to the vector. Secondly, a pair of SapI recognition sequences were added to assist cloning of the GOI variant library. In the N-terminal vector, the SapI sites are located between the linker sequences and IRES. For the C-terminal vector, the SapI sites are located between the Kozake site and the linker sequence of EGFP.
Barcodes and GOI Design
Barcode sequence design:
Below is the final amplicon sequence for adding barcodes to either N-& C- terminal plasmids.
The Italicized template sequence is a double-stranded DNA fragment. BsmBi/EspsI sequences (in red) are added to both ends of the template by a primer set (in bold) using PCR amplification. The lowercase indicates fragment-specific overhangs for backbone assembly. In particular, a degenerative reverse primer has designed a 18nt bases (N) barcode. The lowercase indicates fragment-specific overhangs for backbone assembly. The underlined sequence is important for downstream genomic sequencing.
Gene of Interest flanking design:
VAMP-seq v2 vectors have two pre-existing SapI sites and pre-selected fragment specific overhangs ready to receive GOI sequence with complimentary flasking ends. The specific flasking sequences are as below:
In the N-terminus system, 5’-atg and 5’-CTA (reverse complement of 5’-tag”) are the pre-selected fragment-specific overhangs in the backbone. The insert fragment must contain the SapI recognition sequences (in red) and overhangs (indicated as lowercase) as the following:
In the C-terminus system: 5’-atg and 5’-CGT (reverse complement of 5’-acg) are the pre-selected fragment-specific overhangs in the backbone to receive a compatible insert fragment. The insert fragment must contain the SapI recognition sequences (in red) and overhangs (indicated as lowercase) as the following:
*A stop codon should not be placed at the end of GOI sequence when assembling to the C-terminus backbone. It is already placed at the end of the EGFP. Albeit, in the N-terminus system Albeit, the stop codon is necessary for the GOI insert. It is a part of the SapI overhang.
Both Start and Stop codons cannot be mutated.
Wild-type and destabilizing variants design:
Wild-type of Gene of Interest:
Wild type sequences for gene of interest(s) can be obtained from PCR amplification of existing template, or synthesized as double-stranded DNA fragments. In both cases, the interior sequence should be free of SapI recognition sequences and flanked by the above sequences suitable for the desired designation vectors.
