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Construction of efficient expression vector system using cis-acting elements for Lactococcus lactis

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dc.description학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2014. 2. 최윤재.-
dc.description.abstractMany approaches have been attempted to improve the heterologous protein in various bacteria. Specifically, the plasmid-based expression system has been used to achieve the recombinant protein production as an easy and useful tool to manipulate. There are three strategies to improve the expression level of recombinant protein. Such as 1) introduction of high copies plasmid-based expression vector system, 2) construction of gene multimerization cassette as a insert and ligation with the backbone vector, and 3) search of new strong promoter. However there are several limitations for these strategies in that it is hard to replicate the DNA, is too large to transformed which causes genetic instability, and is hard to predict promoter strength. In this study, I modified the promoter region and tested on the promoter strength. In addition, I tried to introduce another cis-acting elements such as a transcriptional terminator and RBS (ribosome binding site) to improve the expression of recombinant protein.
Lactococcus lactis subsp. lactis IL1403 is widely used in the dairy and animal industries, and it is also studied for a live oral vaccine product to elicit mucosal immune response. The translational elongation factor Tu (tuf) gene is a house-keeping gene, and tuf promoter is characterized as a strong promoter in IL1403. In this study, tuf promoter was modified to test the efficiency of protein expression using the luciferase gene as a reporter.
Firstly two terminators, TrrnB and TpepN were tested for the luciferase gene expression efficiency. TpepN terminator showed better performance in luciferase expression.
Next, series of tuf promoter modification were attempted. In bacteria, RNA polymerases and several sigma factors recognized and recruited approximately -35 and -10 region upstream from the transcription start site. The core region including -35 and -10 hexamers in tuf promoter (119 bp) was amplified and series of modified tuf promoters were constructed using PCR with partial complementary reverse primer. There PCR products (#1) were cloned into the promoterless pIL.Ptuf.Luc(X) vector. Luciferase activity of t2, t4, t6 and t7 were higher than control tuf promoter. Especially t2 and t4 showed better performance, thus selected for next experiment.
It is well known that the sequence between RBS and start codon (ATG) are important for protein translation efficiency. Thus, I modified original sequence of this region, 'CATTTTTCAT' to 'AATTTTTAAA' to give more AT-rich. This modification was combined with selected modified tuf promoter to give a series of new tuf promoter cassette. The transformed IL1403s containing modified promoter (#2) were assayed for luciferase activity. Derivative of t2 and t4, t2-1 and t4-1 showed better performance.
Combined all the modified clones, luciferase activity was compared. t4-1 showed much higher activity compared to the t4, indicating the sequence between RBS and start codon is important for protein expression. To confirm this results, luciferase expression was analyzed on SDS-PAGE and western blot assay. Luciferase bands (61 kDa) was not detectable in SDS-PAGE, but in western blot, clones with t2, t4, t4-1 showed stronger signal compared to original tuf one.
In conclusion, this study revealed that introduction of modified strong promoter and additional cis-acting elements can improve the protein expression in IL1403. And this strategy has a prospect to improve recombinant protein expression. Since pIL252 is a low copy plasmid-based expression system, high copies-based plasmids are needed to increase recombinant protein expression.
dc.description.tableofcontentsSummary I
Contents IV
List of Tables and Figures VII
List of Abbreviations IⅩ

I. Introduction 1

II. Review of Literature 3
1. Lactic Acid Bacteria (LAB) 3
1) Lactic Acid Bacteria 3
2) LAB as probiotics 4
3) Lactococcus lactis 7
2. Recombinant protein expression 8
1) Plasmid-based expression system 8
2) Recombinant protein expression strategy 8
3. Cis-acting elements 9
1) Promoter 9
2) Cis-acting elements 10
4. The tuf gene 11

III. Materials and Methods 12
1. Bacterial cultivation 12
1) Culture medium 12
2) Cultivation and harvest of bacterial cells 12
2. Transformation of bacteria 13
1) Preparation of IL1403 competent cells 13
2) Transformation of bacterial cells 13
3. DNA works 14
1) Plasmid 14
2) Preparation of plasmid DNA 14
3) Enzyme treatment 17
4) PCR reaction 17
5) PCR purification 17
6) Analysis of nucleotide sequences 18
4. Luciferase assay 20
1) Growth phase-dependent luciferase expression 20
2) Detection of chemiluminescence 20
5. Protein works 21
1) Protein extraction from LAB cells 21
2) Quantification of proteins 21
3) SDS-PAGE and western blot assay 21
4) Intensity measurement of protein bands 22
6. In vitro characterization 23
1) Growth of LAB cells 23
2) pH measurement of LAB cultures 23

IV. Results and Discussion 24
1. Introduction of reporter gene with terminator 24
1) Cloning of luciferase gene 24
2) Vector construction 25
2. Validation of reporter gene and terminator 26
1) Growth characteristics 26
2) Luciferase assay 27
3. Modification of tuf promoter (#1) 28
1) Amplification of short fragments 28
2) Construction of repeated fragments 29
3) Elongation of ribosome binding site (RBS) and enzyme sites 30
4) Introduction of modified promoter (#1) 31
4. Modified promoter (#1) activity assay 35
1) Growth characteristics (#1) 35
2) Luciferase assay (#1) 35
5. Construction of remodified promoter (#2) 37
1) Core promoter cassette preparation from modified promoter (#1) 37
2) Elongation of RBS and modified downstream sequences 38
6. Remodified promoter (#2) activity assay 41
1) Luciferase assay (#2) 41
2) Final luciferase assay 42
7. Confirmation of Protein expression 44
1) SDS-PAGE and western blot assay 44

V. Literature Cited 46

VI. Summary in Korean 58

VII. Acknowledgement 61
dc.format.extent1343495 bytes-
dc.publisher서울대학교 대학원-
dc.subjectLactic acid bacteria-
dc.subjectcis-acting element-
dc.subjectpromoter modification-
dc.titleConstruction of efficient expression vector system using cis-acting elements for Lactococcus lactis-
dc.contributor.AlternativeAuthorKim Inseon-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
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