Overexpression of eGFP in Bacillus subtilis DB104 using cry2Aa and cyt1Aa promoters

다국어 초록 (Multilingual Abstract)

Strong promoters for overexpression of recombinant proteins in Bacillus subtilis have been developed. In this study, the cry2Aa promoter (Pcry2Aa) and cyt1Aa promoter (Pcyt1Aa), which are known to be strongly expressed in B. thuringiensis, were introduced into B. subtilis DB104. Pcry2Aa was replaced with a σE consensus promoter sequence, and the three Shine-Dalgarno (SD) of the cry2Aa operon were individually compared. A dual promoter was constructed cyt1Aa Bt II promoter and cry2Aa promoter (Pcyt1Aa-Pcry2Aa) with SD sequence of the cry2Aa structure gene (SD3). The protein expression ability of each promoter was compared using the eGFP reporter gene. As a result, when the σE consensus promoter sequence was applied to -35 promoter of Pcry2Aa, protein expression was increased 4.09-fold. Among the SD sequences constituting the cry2Aa operon, SD3 showed the highest protein expression ability. However, the σE consensus promoter sequence and SD3 were simultaneously applied, a synergistic effect was not observed. In case of the dual promoter, the fluorescence intensity increased 10.71-fold. Therefore, these results suggest that the engineered promoter using cry2Aa and cyt1aa promoter can be applied for recombinant protein production.

국문 초록 (Abstract)

Bacillus subtilis를 활용한 recombinant protein 생산을 위해 다양한 engineered promoter가 개발되어 왔다. 본 연구에서는 B. thuringiensis에서 강하게 발현되는 것으로 알려진 cry2Aa promoter (Pcry2Aa)와 cyt1Aa promoter (Pcyt1Aa)를 B. subtilis DB104에 도입하고, eGFP의 형광측정을 통해 promoter engineering에 의한 단백질 발현량를 비교한다. Pcry2Aa를 B. subtilis에서 sigma E dependent promoter에 대한 consensus sequence로 대체하였고, cry2Aa operon을 구성하는 3개의 Shine-Dalgarno (SD)서열을 개별적으로 비교하였다. 결과적으로 Pcry2Aa의 -35 서열을 변형하였을 때 4.09배, cry2Aa structure gene 발현을 조절하는 SD서열을 독립적으로 적용하였을 때 6.67배 promoter activity가 증가하였다. 하지만 변형된 -35 promoter sequence에 cry2Aa structure gene 발현을 조절하는 SD서열을 함께 적용한 경우, eGFP 발현이 감소하였다. 따라서 dual promoter 구성은 cyt1Aa의 Bt II promoter와 cry2Aa promoter에 (Pcyt1Aa-Pcry2Aa) cry2Aa structure gene SD서열을 함께 적용하였으며, 이 때 형광세기는 10.71배 증가하여 가장 높은 promoter activity를 나타냈다. 따라서 cry2Aa와 cyt1Aa의 promoter를 이용하여 B. subtilis DB104에서 recombinant protein overexpression system 개발 가능성을 확인하였다.

목차 (Table of Contents)

• Ⅰ. Introduction 1
• Ⅱ. Materials and Methods 4
• 2.1. Bacterial strains, plasmids and growth conditions 4
• 2.2. Recombinant DNA techniques 4
• 2.3. Construction of plasmids 4
• 2.4. Measurement of eGFP expression 5
• 2.5. RNA stability 6
• 2.5.1. Total RNA extraction 6
• 2.5.2. Reverse Transcription 7
• 2.5.3. Quantitative real-time PCR (qPCR) 7
• Ⅲ. Results 12
• 3.1. Protein expression ability of cry2Aa wild-type promoter 12
• 3.2. Core promoter sequence substitution of cry2Aa operon 12
• 3.3. Selection and evaluation of SD sequences in cry2Aa operon 15
• 3.4. Application of the σE consensus promoter sequence and optimal SD sequence in cry2Aa promoter 18
• 3.5. Dual promoter 20
• 3.6. RNA half-life 22
• 3.7. Data analysis 24
• Ⅳ. Conclusion 29
• 국문초록 31
• Reference 33