Ali, Muhamad and Yunita Sabrina, Yunita and Amin, Muhamad Modification of Nucleotide Sequences at Upstream and Downstream of the Shine-Dalgarno Sequence to increase DNA Expression in Cell Free System. In: The 2 nd International Conference on Science and Technology 2017 “Joint International Conference on Science and Technology in The Tropic”, 23-24 August 2017, Mataram.
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Abstract
This research aimed at investigating the effect of nucleotide-sequence modification of upstream and downstream at the Shine-Dalgarno sequence on the yield of DNA expression in an Escherichia coli cell-free expression system. An enzyme derived from E.coli, Chloramphenicol Acetyltransferase (CAT), was used as a reporter protein. A fragment of T7 promoter (T7P) was amplified from pK7.CAT plasmid using T7P forward (T7Pf) and three homotail T7P reverse primers (T7Pr-A1, T7Pr-A2, T7Pr-A3). The primers contained different sequences of upstream and downstream at the Shine-Dalgarno (SD) sequence to generate three T7P fragments. Then, three CAT fragments containing a different homotail sequence complementary to the upstream and downstream of the SD sequence were generated using three homotail primers (A1-CAT, A2-CAT, A3-CAT). Three expression cassettes (T7P-A1-CAT, T7P-A2-CAT, T7P-A3-CAT) were constructed and expressed in E. coli cell-free expression system using an overlapping PCR technology. The expression of targeted protein was determined using a CAT assay without purification. Result of this experiment showed that the expression level of the reporter protein was highly dependent on the sequence upstream and downstream of the SD sequence. This result suggest that insertion of CCTCTTT sequence in upstream region and GATTAGACA sequence in downstream part of SD sequence is a potential technique to improve expression level of recombinant protein in the cell-free expression system.
| Item Type: | Conference or Workshop Item (Paper) |
|---|---|
| Keywords (Kata Kunci): | DNA expression, Shine-Dalgarno sequence, Chloramphenicol Acetyltransferase (CAT), cell-free expression system, proteomics assay |
| Subjects: | R Medicine > R Medicine (General) S Agriculture > SF Animal culture |
| Divisions: | Fakultas Kedokteran Fakultas Peternakan |
| Depositing User: | . Zulkarnain Zulkarnain |
| Date Deposited: | 06 Nov 2020 08:23 |
| Last Modified: | 06 Nov 2020 08:23 |
| URI: | http://eprints.unram.ac.id/id/eprint/18241 |
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