• 농업과학연구
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• 제49권4호
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• pp.795-807
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• 2022

The development and commercialization of industrial genetically modified (GM) organisms is actively progressing worldwide, highlighting an increased need for improved safety management protocols. We sought to establish an environmental monitoring method, using real-time polymerase chain reaction (PCR) and propidium monoazide (PMA) treatment to develop a quantitative detection protocol for living GM microorganisms. We developed a duplex TaqMan quantitative PCR (qPCR) assay to simultaneously detect the selectable antibiotic gene, ampicillin (AmpR), and the single-copy Escherichia coli taxon-specific gene, D-1-deoxyxylulose 5-phosphate synthase (dxs), using a direct cell suspension culture. We identified viable engineered E. coli cells by performing qPCR on PMA-treated cells. The theoretical cell density (true copy numbers) calculated from mean quantification cycle (Cq) values of PMA-qPCR showed a bias of 7.71% from the colony-forming unit (CFU), which was within ±25% of the acceptance criteria of the European Network of GMO Laboratories (ENGL). PMA-qPCR to detect AmpR and dxs was highly sensitive and was able to detect target genes from a 10,000-fold (10^-4) diluted cell suspension, with a limit of detection at 95% confidence (LOD_95%) of 134 viable E. coli cells. Compared to DNA-based qPCR methods, the cell suspension direct PMA-qPCR analysis provides reliable results and is a quick and accurate method to monitor living GM E. coli cells that can potentially be released into the environment.

• Journal of Microbiology and Biotechnology
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• 제11권2호
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• pp.281-286
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• 2001

Since the number of amino groups which are exposed by deacetylation of acetyl-D-glucosamine influences antimicrobial activity, a chitosan oligosaccharide (COS) derivative by N-conjugation of COS with asparagine, an amino acid with two amino groups, was synthesized and the antimicrobial effect on E. coli growth was compared with other COS derivatives which were N-conjugated with glycine, alanine, aspartic acid, cysteins, an methionine, and unmodified COS. The structure of asparagine N-conjugated COS (Asn-COS) derivative was identified by using a FT-IR, $^{13}C\;FT-NMR$, and an elemental analyzer. The antimicrobial activity of Asn-COS against E. coli growth was significantly improved as compared to the other COS derivatives as well as COS itself. This means that Asn-COS with two positive charges strongly interacts with the carboxyl negative charges on the bacteria cell wall. The results for Asn-COS were as follows: 100% bactericidal activity, 0.002% MIC, and no growth of E. coli during 3 days of culture time, suggesting that Asn-COS may be useful as a new antibiotic agent.

• 한국미생물·생명공학회지
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• 제23권6호
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• pp.659-664
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• 1995

To improve stability of recombinant DNA pLC1 encoding endoglucanase gene and pGL1 encoding $\beta $-glucosidase gene, DNA fragments of genes coding endoglucanase and $\beta $-glucosidase were cloned within the recA gene on a pDR1453, and the pDRE10 and pDRG20 of recombinant plasmids were integrated into the recA gene on the E. coli 1100 chromosomal DNAs. The stability of inheritance was completely maintained in E. coli 1100; Transformants E. coli 1100/pDREIO and pDRG20 were expressed well by recA promoter and increased endoglucanase and $\beta $-glucosidase activities. This method can be used as a model to improve the stability of recombinant plasmid in large scale culture.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권4호
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• pp.274-277
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• 2004

Production of green fluorescent protein (GFP) as a model foreign protein using different culture scales under co-expression of Vitreoscilla hemoglobin (VHb) in the industrial Escherichia coli strain W3110 (a K12 derivative), was examined. It was found that the VHb co-expressing W3110, exhibited an exceptional and sustained production ability during cell cultures using different scales, while the VHb non-expressing strain showed variable production levels. This high and sustained production ability indicates that the VHb co-expressing E. coli W3110, could be successfully employed for practical large-scale production cultures without the need for serious consideration of scale-up problems.

• 한국미생물·생명공학회지
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• 제32권2호
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• pp.149-153
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• 2004

븐 연구에서는 E. coli MT201을 이용한 L-threonine 발효공정을 확립하였다. 회분식 배양에서 L-threonine생산이 균체 증식과 더불어 증가하는 증식과 연관된 형태의 생산 곡선을 보여주었다. L-Threonine 생산에 대한 조건을 확립하기 위한 유가식 배양에서 첨가배지내 최적 효모추출물과 포도당의 농도는 각각 60 g/$\ell$와 600 g/$\ell$이였으며, 약 87 g/$\ell$의 L-threonine이 생산되었다. 유가식 배양에서도 L-threonine 생산은 회분식 배양과 마찬가지로 균체량의 증가와 더불어 L-threonine 생산이 증가하는 증식과 연관된 형태를 보여주었다. 이러한 결과를 바탕으로 적절한 균체량의 증가와 L-threonine 생산성 향상을 위하여 L-methionine과 인산염이 추가로 공급된 첨가배지를 이용한 고농도 배양 조건이 확립되었다. 한편 고농도 배양에 따른 용존산소의 결핍을 해결하기 위하여 산소가 포함된 혼합공기를 사용하였다. 최적화된 유가식 배양 결과 균체 증식도 원활하게 증가하였으며, L-threonine의 생산도 약 98 g/$\ell$로 향상되었다. 이때 L-threonine의 생산성은 약 3.85 g/$\ell$/h이었다.

• 미생물학회지
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• 제48권3호
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• pp.207-211
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• 2012

이 연구의 목적은 코리네형 균주(Corynebacterium glutamicum)에 존재하는 acyltransferase 유전인자의 발현에 관여하는 autoinducer에 대한 정보를 얻기 위해 다양한 종류의 균주에서 얻은 세포배양액(cell-free culture fluids)을 처리하여 발현에 미치는 효과를 조사하는 것이다. 다양한 배양시간동안 얻은 Agrobacterium tumefaciens의 세포배양액은 acyltransferase의 발현을 전혀 증가시키지 못하는 것으로 관찰되었다. 반면 AI-1과 AI-2를 분비하는 것으로 알려진 Vibrio harveyi BB120($AI-1^+$, $AI-2^+$)을 지수성장기까지 배양시켜 얻은 세포배양액은 acyltransferase의 발현을 증가시키는 것으로 관찰되었다. 이들 autoinducer가 미치는 효과를 조사하기 위하여 돌연변이 균주인 MM77 ($AI-1^-$, $AI-2^-$)과 BB152 ($AI-1^-$, $AI-2^+$)를 사용하여 조사한 결과, 이들 균주에서 얻은 세포배양액은 BB120와는 달리 acyltransferase의 발현을 증가시키지 않는 것으로 나타났다. 이러한 결과로 보아 acyltransferase의 발현은 AI-1에 의한 효과로 보이며, 이를 확인하기 위해 V. harveyi BB152와 같이 AI-2만을 분비하는 것으로 알려진 Escherichia coli ($AI-1^-$, $AI-2^+$)를 사용하였다. 하지만 E. coli 세포배양액은 acyltransferase의 발현을 증가시키는 것으로 관찰되었다. 이들 결과는 코리네형 균주에 존재하는 autoinducer는 기존에 알려진 형태의 신호분자와 차이가 있을 것이며, 코리네형 균주외에 V. harveyi와 E. coli에도 존재하여 종간 상호작용(interspecies communication)에 관여할 것으로 보인다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제38권1호
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• pp.14-19
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• 2012

Introduction: This study was conducted to evaluate ssrA expression resulting from adaptation of Escherichia coli (E. coli) to oral pathogens through signal exchange. Materials and Methods: Human cell lines Hep2 and HT29, wild-type E. coli (WT K-12), ssrA knock-out E. coli (${\Delta}K$-12), and Scleropages aureus (S. aureus) were used. A single culture consisting of Hep2, HT29, WT K-12, and ${\Delta}K$-12, and mixed cultures consisting of Hep2 and WT K-12, Hep2 and ${\Delta}K$-12, WT K-12 and S. aureus, ${\Delta}K$-12 and S. aureus, and Hep2, WT K-12, and S. aureus were prepared. For HT29, a mixed culture was prepared with WT K-12 and with WT K-12 and S. aureus. Total RNA was extracted from each culture with the resulting expression of ssrA, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-${\kappa}B$), and p53 was evaluated by Reverse transcription polymerase chain reaction (RT-PCR). Results: The expression of ssrA in a single culture of WT K-12 was lower than that observed in the mixed culture of WT K-12 with S. aureus. Greater ssrA expression was observed in the mixed culture of WT K-12 with Hep2 than in the single culture of WT K-12. The expression of NF-${\kappa}B$ was higher in the mixed culture of Hep2 with ${\Delta}K$-12 than that in the mixed culture of Hep2 with WT K-12, and was lowest in the single culture of Hep2. The expression of ssrA was higher in the mixed culture of WT K-12 with Hep2 and S. aureus than in the mixed culture of WT K-12 with Hep2. Conclusion: These results suggest that ssrA plays an important role in the mechanism of E. coli adaptation to a new environment.

• KSBB Journal
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• 제11권4호
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• pp.389-397
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• 1996

인산암모늄의 첨가에 따른 재조합 대장균의 성장과 그 부산물 생성 관계를 알기 위하여 인산암모늄의 청가 상태에 따른 영향을 검토하였다. 인산암모 늄은 세포 성장에 중요한 작용을 하고 있다. 배양 실시후 8시간에서 12시간 전후에서 인산 암모늄이 거의 흡수되었고 탄소원이 세포에 흡수되는 속도와 인산암모늄이 세포에 흉수되는 속도가 거의 비슷한 현 상으로 진행되었다. 인산 암모늄의 초기농도를 O.5g/ L로 사용할 때 세포 최대 농도는 4.2g/L로 최고를 보였다. 그러으로 세포 성장에 펼요한 인산 암모늄 의 첨가를 초기 인산암모늄 농도 O.5g/L 전후로 첨가함이 적당하다. 그리고 인산암모늄에 trypton을 첨가하지 않는 LB 배지와 trypton을 첨가할 LB 배지에서 세포 성장은 비슷한 결과로 성장되었으며 부산물들의 생성은 acetic acid의 경우는 Trypton을 첨가하지 않으면 많이 생성되고 lactic acid의 경우 는 trypton을 첨가할 때 매우 높게 축적되어졌다. 그러므로 trypton은 세포성장에 그렇게 중요하지 않 는 결과를 보였으며 저해제인 부산물을 적게 나오게 하려면 trypton을 첨가함이 좋고 lactic acid 생성 관계는 더욱 연구되어져야 한다.

• Journal of Microbiology and Biotechnology
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• 제8권3호
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• pp.245-250
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• 1998

A gene encoding an extracellular collagenase from the marine bacterium Vibrio vulnificus CYK279H was cloned into E. coli JM83 using the multicopy plasmid vector pUC19. The cloned strain of recombinant E. coli showing collagenase activity had an insert fragment of 3.5 kb and was named E. coli JM83/pKCL 279H. The cloned strain produced two different collagenase during cultivation. These enzymes, named collagenase-I and -II, were purified from the culture supernatant. SDS-PAGE indicated that collagenase-I had a molecular weight of 41 kDa and collagenase-II had a weight of 37 kDa. The N-terminal amino acid sequence of collagenase-I from the cloned strain, E. coli JM83/pKCL279H was determined and was not found to be similar to any other known collagenases. The optimum pH and temperature of the purified collagenase-I were 7.8 and $37^{\circ}C$, respectively.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.777-782
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• 2004

The arylsulfatase gene (astA, 984 bp ORF) from the P. carrageenovora genome was amplified by PCR and subcloned into the pET21a vector. When the constructed plasmid pAST-A1 (6.4 kb) was introduced into E. coli BL21(DE3), the transformant on the LB plate containing IPTG showed a hydrolyzing activity for 4-methylumbelliferyl sulfate and p-nitrophenyl sulfate. The highest arylsulfatase activity (2.1 unit/ml) was obtained at 10 mM IPTG. Most arylsulfatase activity was found in the cell lysate, whereas no significant activity was detected in the culture supernatant. The molecular weight of the recombinant enzyme was estimated to be 33.1 kDa by SDS-PAGE. After the reaction of agar with arylsulfatase for 12 h at $40^{\circ}C$, the gel strength of the agar increased by 2-fold, and 73% of the sulfate in the agar had been removed. This result suggests that arylsulfatase expressed in E. coli could be useful in the production of electrophoretic grade agarose.