• IMMUNE NETWORK
• /
• 제9권6호
• /
• pp.243-247
• /
• 2009

In this study, we report the development of a new dual reporter vector system for the analysis of promoter activity. This system employs green fluorescence emitting protein, EGFP, as a reporter, and uses red fluorescence emitting protein, DsRed, as a transfection control in a single vector. The expression of those two proteins can be readily detected via flow cytometry in a single analysis, with no need for any further manipulation after transfection. As this system allows for the simultaneous detection of both the control and reporter proteins in the same cells, only transfected cells which express the control protein, DsRed, can be subjected to promoter activity analysis, via the gating out of all un-transfected cells. This results in a dramatic increase in the promoter activity detection sensitivity. This novel reporter vector system should prove to be a simple and efficient method for the analysis of promoter activity.

• Journal of Microbiology and Biotechnology
• /
• 제31권6호
• /
• pp.833-839
• /
• 2021

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (P_cry3A, P₁₀, P_SG1, P_srfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P₁₀ promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2001년도 추계학술발표대회
• /
• pp.762-766
• /
• 2001

The nar promoter of Escherichia coli was known to induce maximally under anaerobic or microaerobic conditions in the presence of nitrate. In this study, the nar promoter was tested to see whether the expression level of a reporter gene which fused lacZ gene at nar promoter's downstream, in the some gram negative host strains(Agrobacterium, Pseudomonas and Rhizobium). A nar promoter system(Combination of nar promoter and gram negative strain) was grown under aerobic conditions to absorbance at 600 nm of nearly 2.0 and then, the nar promoter was induced by lowering DO to 1-2% with alternating microaerobic and aerobic condition in the fermentor cultures, using different gram negative hosts. For a wild type nar promoter (pNW61), it was possible to maintain production of ${\beta}-galactosidase$ activity per cell(specific ${\beta}-galactosidase$ activity) at 14,000, 9600, 45 Miller units in the presence of 1% nitrate. and for a nitrate - independent nar promoter (pNW618) at 12,000, 10,400 and 58 Miller units in the absence of nitrate ion, respectively.

• Journal of Plant Biology
• /
• 제37권3호
• /
• pp.371-380
• /
• 1994

To determine the patterns and the levels of expression of the cauliflower mosaic virus (CaMV 35S) promoter and the rice actin 1 (Act1) promoter in rice, transgenic rice plants containing CaMV 35S-$\beta$-glucuronidase (GUS) and Act1-GUS constructs were generated and examined by fluorometric and histochemical analyses. The fluorometric analysis of stably transformed calluses showed that the activity of the rice Act1 promoter was stronger than that of the CaMV 35S promoter in rice cells. In a histochemcial study of the transgenic rices, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in parenchymal cells of vascular tissues of leaves and roots and mesophyll cells of leaves. These results are similar to those of potato, a dicot plant. In contrast, rice plant transformed with Act1-GUS fusion construct revealed strong GUS activity in parenchymal cells of vascular tissue, mesophyll cells, epidermal cells, bulliform cells, guard subsidiary cells of leaves and most cells of the root, suggesting that the rice Act1 promoter is more constitutive than the CaMV 35S promoter. It was also confirmed that in both types of transgenic rice little or no staining was localized in metaxylen tracheary elements of vascular tissue from leaves or roots. These results indicate that the rice Act1 promoter can be utilized more successfully for expression of a variety of foreign gene in rice than the CaMV 35S promoter.

• 한국미생물·생명공학회지
• /
• 제16권5호
• /
• pp.343-347
• /
• 1988

토양에서 분리한 알칼리 내성 Bacillus속의 chromosomal DNA로부터 promoter를 cloning하여 선별된 재조합 plasmid p-12내 의 promoter를 subcloning을 하였다. 그 결과 cloning된 promoter 내에는 서로 다른 두 가지의 promoter가 존재하는 것을 확인할 수 있었고 이로부터 각각의 promoter를 함유한 재조합 plasmid p-l2B1, p-l2B2를 제조하였다. 또한 CAT 비활성 측정에 의해 각 promoter의 활성을 비교해 본 결과 p-l2B1의 promoter는 p-l2B2의 promoter에 비해 상대적으로 높은 활성을 가지고 있었다. CAT 비활성을 생육시기에 따라 측정해 본 결과 p-l2B1과 p-l2B2는 대수증식기 이후 활성이 급증되었으며 배지 중 첨가된 1.0%의 glucose에 의해 활성이 억제되는 효과를 받았다.

• 한국미생물·생명공학회지
• /
• 제19권1호
• /
• pp.21-24
• /
• 1991

알칼리 내성 Bacillus sp. YA-14의 chromosomal DNA로부터 분리된 promoter를 subcloning하여 생화학적 특성을 조사하였다. B.subtilis와 promoter 공여균주, Bacillus sp. YA-14에서 promoter의 활성은 포자형성 초기단계에서 급격히 증가하였으며 1.0(w/v) glucose 첨가로 promoter 활성이 억제되었고 c-GMP에 의해 저해되었던 활성이 증가하였다.

• 한국미생물·생명공학회지
• /
• 제18권4호
• /
• pp.406-410
• /
• 1990

토양에서 분리한 알칼리내성 Bacillus sp.의 자체 chromosomal DNA에서 유래된 strong promoter를 지닌 plasmid p-12B1을 공여균주에 삽입시키고 이 promoter의 활성을 최적화할 수 있는 배양조건을 검토하였다. 글루코오스가 고갈된 시점부터 세포의 활성은 유지되나 포자는 형성되지 않을 정도로 낮은 글루코오스 소비속도를 유지해 제한된 글루코오스를 연속적으로 공급해줌으로써 promoter의 활성을 최대로 높일 수 있었다. 또한 배지조성의 변화로 균체를 생육시킨 후 유전자 발현을 유도하는 것이 가능하였으며, 이 점에 대해서는 보다 구체적인 연구가 진행되어야 할 것이다.

• Journal of Plant Biology
• /
• 제38권3호
• /
• pp.259-266
• /
• 1995

To investigate how the dyad symmetry region (DSR) and the distance between vir box and -35 sequence of the virE promoter plays a role in virE gene expression, two mutants were constructed by base substitution and insertional mutagenesis. The base substitutional mutation, a AAlongrightarrowCG substitution at positions -39 and -40 on the DSR, showed the level of $\beta$-galactosidase activity approximately 91% of the wild type virE promoter activity. Therefore, the native structure of the DSR seems to be not essential for virE expression. The insertional mutation, constructed by inserting 8 bp ClaI linker between -49 and -50, displayed the $\beta$-galactosidase activity at 12% of the native virE promoter activity. However, this striking reduction appears to be not caused by destruction of the native DSR structure, but by shifting the vir box far from putative -35 sequence.

• Journal of Microbiology and Biotechnology
• /
• 제15권3호
• /
• pp.678-682
• /
• 2005

The promoter region of a carbon starvation gene isolated from Pseudomonas putida was cloned and analyzed for its potential use for in situ bioremediation and bioprocessing. We constructed a recombinant plasmid pMKD101 by cloning the 0.65 kb promoter region of the gene into the promoter proving vector, pMK301, which contains the lacZ for ${\beta}$-galactosidase activity as a reporter gene. pMKD101 was transformed into the wild-type P. putida MK1, resulting in P. putida RPD101, and analyzed for ${\beta}$-galactosidase activity under different culture conditions. When RPD101 was grown on the minimal medium plus $0.1\%$ glucose as a sole carbon source in batch cultures, ${\beta}$-galactosidase activity was found to be 3.2-fold higher during the stationary phase than during the exponential phase. In chemostat cultures, ${\beta}$-galactosidase activity was found to be 3.1-fold higher at the minimal growth rate (dilution rate=$0.05\;h^{-1}$) than at the maximal growth rate (dilution rate=$0.173;h^{-1}$). The results suggest that a carbon starvation promoter can be utilized to maximize the expression of a desired gene under nutrient limitation.

• Journal of Plant Biology
• /
• 제36권3호
• /
• pp.267-273
• /
• 1993

Promoters of the chlorophyll a/b bidning protein genes, cab1, and cab2, of Arabidopsis thaliana were studied for their functions in differential expression during greening of etiolated shoots. The etiolated shoots were derived from leaves of transgenic tobacco plants with the cab-CAT (chloramphenicol acetyltransferase) translational fusions, and CAT activity was measured to monitor the activities of the cab promoters. Cab1 promoter activity increased rapidly and showed saturation after about 24 hours of greening, but that of cab2 increased with about 2 day-lag period and showed saturation after 6 days. Cab1 promoter activity was more sensitive to levulinic acid (LA) compared with cab2 activity. Cab2 promoter activity was inhibited more sensitively by chloramphynicol (CAP) than by inhibitors of Chl formation. Cab1 promoter activity was, however, inhibited less sensitively by CAP than by LA. The treatment of abscisic acid (ABA) did not block Chl synthesis so significantly as LA treatment did, and cab2 promoter activity was much less sensitive to ABA compared with that of cab1. These results suggest that cab1 expression is strongly related with Chl formation, possibly with $\delta$-aminolevulinic acid accumulation, and cab2 expression is suppressed more by the blockage of translation of Chl a-apoproteins than by the blockage of Chl a accumulation.