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Faculty Mentor

Fedora Sutton

Abstract

HVCR21 is a barley protein known to be cold regulated at the mRNA level. However, its function as well as the affect of low temperature on its translation are unknown. The purpose of this project was to engineer E. coli to express recombinant HVCR21. PGR primers were designed for the 5' and 3' ends of the HVCR21 coding region. The primers were also designed with a 5' SacI restriction site and a 3' Pst I restriction site. After ligation of the PCR product into the pCR4-TOPO vector, bacteria were transformed and plated and the successful transformant verified by PCR. The new pCR4-TOPO-HVCR21 construct was therefore available as a ready source of the SacI/Pst I fragment. The expression vector pQE100 was prepared by digestion with SacI and Pstl. The Sacl/Pst I fragment from pCR4-TOPO-HVCR21 was ligated to the SacI/PstI pQE100 vector. Transformants were analyzed by PCR with the HVCR21 primers. Sequence analysis of the engineered region of pQE100-HVGR21 confirms the correct orientation and frame for expression of HVCR21 in E. coli. Future studies will involve growth of bacteria containing this construct and SDS-polyacrylamide gel electrophoresis analysis of the expressed proteins.

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