가뭄 스트레스 특이적인 cis-regulatory element의 특성을 기반으로 한 신규 프로모터 구축

김기환 ( Kihwan Kim ),김병규 ( Byeonggyu Kim ),신주형 ( Juhyung Shin ),김원찬 ( Won-chan Kim ) 한국응용생명화학회 2021 Journal of Applied Biological Chemistry (J. Appl. Vol.64 No.1

Droughts are one of the abiotic stresses that hinders the growth and productivity of crop plants. Coping with abiotic stress is necessary to understand the molecular regulatory networks that makes plants respond to adverse environmental conditions. In our experiment to find a combination that can cope with abiotic stress (respond to drought), we screened 5 stressinducible promoters that are expressed only under stress conditions. This founded 36 cis-elements in stress-inducible promoters. With the result we designed 2 synthetic promoters (BL1, BL2) for fine-controlled regulation by assembling ciselements from the native promoters, which are expressed only under stress caused by droughts. Analysis of the transgenic plant (BL1-GUS, BL2-GUS) showed that the synthetic promoters increased the expression of β-glucuronidase (GUS) in transgenic plants under desiccation. Also in the transient activation assay demonstrated that synthetic promoters induced the cotransformation of effector DREB1A and DREB2C. These results expect that the synthetic promoter with a combination of droughtspecific elements can be used to respond to various abiotic stress and is resistant to stress without causing growth retardation.

Promoter-optimized Cas9 Gene Enables Highly Efficient CRISPR/Cas9-based Genome Engineering in Streptomyces

Hyun-woo JE,Hi-young KIM,Chang-hun JI,Hahk-Soo KANG 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

CRISPR/Cas9 system provides an efficient tool for genome engineering of Streptomyces, a prolific source of commercially valuable secondary metabolites. However, the application of CRISPR-Cas9 system to Streptomyces strains has been limited due to the toxicity associated with Cas9 protein, resulting in low transformation efficiency or failure of genome engineering. Here, we engineer the Cas9 gene promoter to optimize its expression with an aim to reduce toxicity while maintaining high engineering efficiency. Randomized Cas9 promoters were screened using our indigoidine synthetase-based reporter system that allows the selection of genome-engineered positive transformants based on color. As a proof of concept, we applied this strategy to three model Streptomyces strains including S. coelicolor, S. albus, and S. roseosporus. The results of promoter activity assay for positive transformants suggest that optimal levels of Cas9 expression varies between strains. We successfully deleted large biosynthetic gene clusters using the CRISPR/Cas 9 plasmids with optimized Cas9 expression in three model Streptomyces strains.

Glucose-responsive artificial promoter-mediated insulin gene transfer improves glucose control in diabetic mice.

Han, Jaeseok,Kim, Eung-Hwi,Choi, Woohyuk,Jun, Hee-Sook WJG Press 2012 WORLD JOURNAL OF GASTROENTEROLOGY Vol.18 No.44

<P>To investigate the effect of insulin gene therapy using a glucose-responsive synthetic promoter in type 2 diabetic obese mice.</P>

Expression Study of a Recombinant Plasmid containing Dipeptidyl Peptidase-4 Gene in E. coli : A Plausible Application for Celiac Disease Patients to Digest Gluten

Yeonjae Lee,Ryan Kang,Jenna Kwon,Kyuhee Jo,Jungbin Im,Sangwook Jung,DongHyun Lee,Juhyeon Lee,Jeong-Sang Lee 한국인터넷방송통신학회 2018 Journal of Advanced Smart Convergence Vol.7 No.2

Celiac disease (CD) is an immune-mediated enteropathy of small intestine diagnosed in both childhood and adulthood. CD is caused by gluten, which produces gliadorphin during its digestion. The enzyme dipeptidyl peptidase-4 (DPP4) breaks gliadorphin down nevertheless the last tripeptide remains and eventually inhibits DPP4, thus slowing down its process. Therefore, the idea is to produce an additional DPP4 enzyme which is crucial. Consequently, the functional DPP4 gene was cloned into pCDNA3 intermediate (FLAG+DPP4) vector and finally a recombinant plasmid pSB1C3 (Andersons promoters+ FLAG+DPP4) was constructed using synthetic biology. Normally, a DPP4 inhibitor is used as a cure for diabetes. Another important concern was overexpression of DPP4, which might lead to diabetes, accordingly the work was also performed for the regulation of the DPP4 gene expression. In this regard, three types of Anderson promoters (strong, moderate and weak) were utilized to study the control overexpression. This is the first report of idealistic trial for control the exogenous DPP4 gene-expression by molecular biologic tools.

Expression Study of a Recombinant Plasmid containing Dipeptidyl Peptidase-4 Gene in E. coli: A Plausible Application for Celiac Disease Patients to Digest Gluten

Lee, Yeonjae,Kang, Ryan,Kwon, Jenna,Jo, Kyuhee,Im, Jungbin,Jung, Sangwook,Lee, DongHyun,Lee, Juhyeon,Lee, Jeong-Sang The Institute of Internet 2018 International journal of advanced smart convergenc Vol.7 No.2

Celiac disease (CD) is an immune-mediated enteropathy of small intestine diagnosed in both childhood and adulthood. CD is caused by gluten, which produces gliadorphin during its digestion. The enzyme dipeptidyl peptidase-4 (DPP4) breaks gliadorphin down nevertheless the last tripeptide remains and eventually inhibits DPP4, thus slowing down its process. Therefore, the idea is to produce an additional DPP4 enzyme which is crucial. Consequently, the functional DPP4 gene was cloned into pCDNA3 intermediate (FLAG+DPP4) vector and finally a recombinant plasmid pSB1C3 (Andersons promoters+FLAG+DPP4) was constructed using synthetic biology. Normally, a DPP4 inhibitor is used as a cure for diabetes. Another important concern was overexpression of DPP4, which might lead to diabetes, accordingly the work was also performed for the regulation of the DPP4 gene expression. In this regard, three types of Anderson promoters (strong, moderate and weak) were utilized to study the control overexpression. This is the first report of idealistic trial for control the exogenous DPP4 gene-expression by molecular biologic tools.

A Rapid and Simple Method for Construction and Expression of a Synthetic Human Growth Hormone Gene in Escherichia coli

Roytrakul, Sittiruk,Eurwilaichitr, Lily,Suprasongsin, Chittiwat,Panyim, Sakol Korean Society for Biochemistry and Molecular Biol 2001 Journal of biochemistry and molecular biology Vol.34 No.6

A cDNA, encoding the human growth hormone (hGH), was synthesized based on the known 191 amino acid sequence. Its codon usage was optimized for a high level expression in Escherichia coli. Unique restriction sites were incorporated throughout the gene to facilitate mutagenesis in further studies. To minimize an initiation translation problem, a 624-bp cassette that contained a ribosome binding site and a start codon were fused to the hGH-coding sequence that was flanked between the EcoRI and HindIII sites. The whole fragment was synthesized by an overlapped extension of eight long synthetic oligonucleotides. The four-short duplexes of DNA, which were first formed by annealing and filling-in with a Klenow fragment, were assembled to form a complete hGH gene. The hGH was cloned and expressed successfully using a pET17b plasmid that contained the T7 promoter. Recombinant hGH yielded as much as 20% of the total cellular proteins. However, the majority of the protein was in the form of insoluble inclusion bodies. N-terminal amino acid sequencing also showed that the hGH produced in E. coli contained formyl-methionine. This study provides a useful model for synthesis of the gene of interest and production of recombinant proteins in E. coli.

Bacterial Hash Function Using DNA-Based XOR Logic Reveals Unexpected Behavior of the LuxR Promoter

Pearson, Brianna,Lau, Kin H.,Allen, Alicia,Barron, James,Cool, Robert,Davis, Kelly,DeLoache, Will,Feeney, Erin,Gordon, Andrew,Igo, John,Lewis, Aaron,Muscalino, Kristi,Parra, Madeline,Penumetcha, Palla Korean Society for Bioinformatics 2011 Interdisciplinary Bio Central (IBC) Vol.3 No.3

Introduction: Hash functions are computer algorithms that protect information and secure transactions. In response to the NIST's "International Call for Hash Function", we developed a biological hash function using the computing capabilities of bacteria. We designed a DNA-based XOR logic gate that allows bacterial colonies arranged in a series on an agar plate to perform hash function calculations. Results and Discussion: In order to provide each colony with adequate time to process inputs and perform XOR logic, we designed and successfully demonstrated a system for time-delayed bacterial growth. Our system is based on the diffusion of ${\ss}$-lactamase, resulting in destruction of ampicillin. Our DNA-based XOR logic gate design is based on the op-position of two promoters. Our results showed that $P_{lux}$ and $P_{OmpC}$ functioned as expected individually, but $P_{lux}$ did not behave as expected in the XOR construct. Our data showed that, contrary to literature reports, the $P_{lux}$ promoter is bidirectional. In the absence of the 3OC6 inducer, the LuxR activator can bind to the $P_{lux}$ promoter and induce backwards transcription. Conclusion and Prospects: Our system of time delayed bacterial growth allows for the successive processing of a bacterial hash function, and is expected to have utility in other synthetic biology applications. While testing our DNA-based XOR logic gate, we uncovered a novel function of $P_{lux}$. In the absence of autoinducer 3OC6, LuxR binds to $P_{lux}$ and activates backwards transcription. This result advances basic research and has important implications for the widespread use of the $P_{lux}$ promoter.

Optimization of Phage λ Promoter Strength for Synthetic Small Regulatory RNA-based Metabolic Engineering

Sung, Minhui,Yoo, Seung Min,Jun, Ren,Lee, Jae Eun,Lee, Sang Yup,Na, Dokyun Korean Society for Biotechnology and Bioengineerin 2016 Biotechnology and Bioprocess Engineering Vol.21 No.4

Synthetic small regulatory RNAs (sRNAs) are gene-silencing tools that can be used to tune gene expression in prokaryotes. A recent study by our group proposed rational design principles, introduced a regulatory system that may be used to implement synthetic sRNAs, and showed their utility in metabolic engineering. The regulatory system employed the strong phage <TEX>${\lambda}$</TEX> <TEX>$P_R$</TEX> promoter to tightly control synthetic sRNA production. Here, we fine-tuned the strength of the <TEX>$P_R$</TEX> promoter via mutagenesis in order to optimize the level of synthetic sRNAs while maintaining the ability of the promoter to be regulated by CI proteins. Five mutant promoters of different strengths, ranging from 24 to 87% of that of the wild-type <TEX>$P_R$</TEX> promoter, were identified and confirmed to be repressed by CI proteins. A mutated promoter with only 40% of the original strength still produced enough synthetic sRNAs to inhibit the translation of the target mRNA to ~10% of the original level. As a practical application, we tested our promoters as drivers for a synthetic anti-murE sRNA, which was used to adjust the production of cadaverine. As the promoter strength decreased, the cadaverine titer first increased and then dropped. A mutated promoter with 39% of the original strength achieved the improved cadaverine titer of 2.15 g/L. The mutant promoters developed in this study should prove useful for tuning the expression levels of synthetic sRNAs for metabolic engineering.

Synthetic Promoter Design for Cyanobacteria with Deep-learning Model

Euijin SEO,Yun-nam CHOI,Ye Rim SHIN,Donghyuk KIM,Jeong Wook LEE 한국생물공학회 2022 한국생물공학회 학술대회 Vol.2022 No.4

연구보문 : 코돈최적화를 통한 합성 조직형 플라스미노겐 활성화인자의 형질전환 담배 내 발현

심준수 ( Joon Soo Sim ),차소영 ( So Young Cha ),한정순 ( Jeong Soon Han ),강솔 ( Sol Kang ),헤마바티 ( He Ma Va Thi ),김용환 ( Yong Hwan Kim ),한범수 ( Bum Soo Hahn ) 한국국제농업개발학회 2010 韓國國際農業開發學會誌 Vol.22 No.4

본 연구에서는 현재 전 세계적으로 혈전용해제로서 널리 사용되어지고 있는 t-PA를 담배에서 생산하기 위해 t-PA 유전자를 코돈최적화를 통해 합성하여 담배 rbcS 프로모터하에서 발현을 유도하였다. 합성된 t-PA 유전자는 발현 벡터를 이용하여 담배 형질전환을 실시하였고, 동물세포 유래 t-PA와의 인공 혈 전용해 활성 및 분자량의 동등성 등의 특성을 규명하였다. 1. 담배의 codon usage에 맞게 t-PA 유전자를 합성하였고, 담배 형질전환체 잎에서 피브린 분해 활성이 있는 효소학적으로 안정된 재조합 t-PA의 발현을 확인하였다. 2. 합성 t-PA 유전자의 담배 genomic DNA내의 삽입과 전사는 PCR과 RT-PCR법으로 측정하였고, t-PA 유전자의 PCR 산물(1.6 kb)을 관찰하여 담배 염색체내에 삽입되어 있음을 확인하였다. 또한 t-PA의 전사체 크기에 해당하는 1.6 kb의 PCR 산물을 관찰하여 담배 잎 내에서 정상적인 전사가 이뤄지고 있음을 확인하였다. 3. 담배 형질전환체 잎의 전체 수용성 단백질 중 합성 t-PA 의 평균 발현양은 0.03 μg/TSP mg으로 측정되었고, 가장 높은 발현양은 0.0903 μg/TSP mg로 관찰하였다. 4. 담배 잎 추출물 내에 존재하는 합성 t-PA 단백질의 피브린 분해 활성은 상업적으로 판매되는 재조합의 t-PA 단백질과 동등한 활성을 나타냈고, 담배 형질전환체들의 피브린 용해 활성의 평균값은 0.2589 cm2 lysis zone을 나타냈고, 피브린 용해 활성의 최대치는 0.9 cm2 lysis zone이었다. 5. 형질전환 담배 잎에서 발현된 재조합 합성 t-PA의 분자량을 분석한 결과 동물세포 유래 t-PA와 유사한 크기의 65 kDa으로 확인하였다. 6. T-PA 단백질을 발현하는 담배 형질전환체들은 식물체의 키, 색깔, 모양, 잎의 개수, 성장속도, 종자의 생산량 등과 같은 표현형에 있어 야생종 담배와 별 다른 차이를 보이지 않았으며 후대에도 안정적인 t-PA 활성을 나타내었다. Human tissue-type plasminogen activator(t-PA) which is found in blood and tissues is one of the proteolytic enzyme. T-PA causes fibrin-specific plasminogen activation that can dissolve blood clots in the vasculature. In order to produce recombinant t-PA(st-PA) proteins in transgenic tobacco plants, the t-PA gene was codon-optimized and cloned into a plant binary vector(p221a-rbcS-st-PA) harboring a tobacco rbcS promoter, a tobacco etch virus leader sequence, an N-terminal signal peptide of the alfala glucose-regulated endoplasmic recticular protein and a 35S terminator. The plasmid was transformed into Agrobacterium tumefaciens EHA105 and then the Agrobacterium-mediated transformation of tobacco leaf discs was performed. The insertion of the st-PA gene in genomic DNA of transgenic tobacco plants and the presence of its transcript were verified by PCR and RT-PCR, respectively. We investigated the fibrinolytic activity of recombinant st-PA proteins and quantified their amount to determine the expression level of st-PA proteins in transgenic tobacco leaves. Enzyme-linked immunosorbent assay determined that the highest level of recombinant st-PA expression was 0.09 μg/total soluble protein(mg) in transgenic tobacco plants. The amount of recombinant st-PA proteins in transgenic plants was estimated to range from 0.001 to 0.009% of total soluble proteins. Immuno-blot analysis of transgenic tobacco leaves revealed a single band of approximately 68 kDa recombinant st-PA proteins. These results demonstrated the expression and in vitro activity of recombinant st-PA in transgenic tobacco plants. Thus it provides the information for the additional production of recombinant pharmaceutical proteins using plant system.