Ubiquitin (UB) cascades in vivo are complex to study with cross-reactivity of E1-E2 pairs, E2-E3 pairs, and E3-substrate pairs. RNF38, RING finger protein 38, is a RING-type E3 ligase in the UB transfer cascade in the cell, and the role of RNF38 in cell regulation is unknown, but one study identifies p53 ubiquitination. Previously, the orthogonal UB transfer (OUT) cascade with xUB~xE1 and xE1~xE2 pairs is used to determine xE2-xE3 pair. The current project focuses on generating RNF38 Ring domain libraries with the randomization of four key residues in the E2-binding site then selecting out specific xE2-xRNF38 RING pairs allowing exclusive transfer of xUB to substrate proteins of RNF38. RNF38 RING library is displayed on the surface of M13 phage for the selection of RING mutants binding with xE2. After identifying functional xE2-xRNF38 pairs, xRNF38 is used to assemble the OUT cascade to identify potential substrates by proteomics.