Screening of Bacterial Recombinants: Strategies and Preventing False Positives

Padmanabhan, Sriram; Banerjee, Sampali; Mandi, Naganath

doi:10.5772/22140

Cited by 14 publications

(16 citation statements)

References 58 publications

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“…As explained by Padmanabhan and collaborators (2016) [33], the molecular mechanism for blue/white screening (that is, recovering of functional β-galactosidase LacZ) is based on a genetic engineering of the lac operon in the E. coli chromosome (coding for the omega peptide with a N-terminal deletion) combined with a subunit complementation achieved with the cloning vector (coding for the α peptide). In this way, tetramerization to produce a functional LacZ enzyme is going to occur only if the α peptide, which correspond to the intact N-terminal portion of the omega peptide, is added in trans [34].…”

Section: Resultsmentioning

confidence: 99%

Big trouble with little inserts: boundaries in metagenomic screenings usinglacZα based vectors

LdF

et al. 2017

Preprint

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21The vast biochemical repertoire found in microbial communities from a wide-range of environments allows screening 22 and isolation of novel enzymes with improved catalytic features. In this sense, metagenomics approaches have been 23 of high relevance for providing enzymes used in diverse industrial applications. For instance, glycosyl hydrolases, 24 which catalyze the hydrolysis of carbohydrates to sugars, are essential for bioethanol production from renewable 25resources. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities 26 from microbial communities inhabiting a soil sample by using the lacZα-based plasmid pSEVA232 in the generation 27 of a screenable metagenomic library. For this, we used a functional screen based on skimmed milk agar and a pH 28 indicator dye as previously reported in literature. Although we effectively identified nine positive clones in the 29 screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded 30 in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within 31 the coding region of the lacZα gene present in the original vector. We concluded that the current method has a higher 32 tendency for false positive clones' recovery, when used in combination with a lacZα-based vector. Finally, we discuss 33 the molecular explanation for positive phenotype recovering and highlight the importance of reporting boundaries in 34 metagenomic screenings methodologies. 35

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Section: Resultsmentioning

confidence: 99%

Big trouble with little inserts: boundaries in metagenomic screenings usinglacZα based vectors

LdF

et al. 2017

Preprint

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“…RNA polymerase reaching this site ceases transcription, so only the cloned gene itself will be transcribed from the strong promoter. [19,20] Furthermore, BL21(DE3) strain also sensitive to the ampicillin while pET-16b vector carry ampicillin resistance as selective marker because of that the addition of ampicillin to the medium and the ability of BL21(DE3) strain grown on it due to harboaring pET-16b plasmid. [21] In this study constructive vector pET-16b-HS was extracted (see Figure 4), and by electrophoresis with control the resulted band of constructive vector about 5,901 bp in comparison with control about 5,711 bp that's mean presence of target gene in constructive vector, for more detection from the constructive vector which extracted from transformed cell, Hirudin genes was amplifying with PCR cycle, the resulted band from gel electrophoresis about 202 bp (see Figure 5).…”

Section: Discussionmentioning

confidence: 99%

Production and purification of constructed recombinant hirudin in BL21(DE3) strain

Al-Badran

Al-Fadal

2017

JBEI

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Background/Objective: Hirudin, an extract from the leech, has powerful antithrombin activity affecting the blood coagulation pathway, it is the most potent natural inhibitor of thrombin, it binds thrombin with high affinity, so, the aim of this study was to build hirudin gene by overlapping extension PCR then cloning and expression in BL21(DE3) strain. Methods: Hirudin gene constructed with four modified primers then the final product amplified by two primers named as A, B by using overlapping extension PCR, for gene expression, BL21(DE3) strain was used under the control of T7 promoter in pET-16b vector and for hirudin production, LB broth medium was used as fermentation medium, to detect the expression of hirudin in BL21(DE3) strain in fermentation media real-time PCR was used. Hirudin protein purified at first by (Immobilized metal affinity chromatography) IMAC, then this protein was dialysis and treated with factor Xa to eliminate His-tag. Then hirudin purified by DEAE sepharose and SP sepharose column, the concentration of protein determined by ELISA, furthermore the activity evaluated by thrombin titration and activated partial thromboplastin time (APPT) test. Results: Hirudin gene constructed in two round PCR first round produced two products (product 1,117 bp while product 2,114 bp) the second-round PCR gave the final product 213 bp. The resulted band from gel electrophoresis for constructed vector pET-16b-HirudinS was (5,901 bp). Hirudin expression was established by real-time PCR with Ct value (20.28). The analysis on 15% SDS-PAGE for the SP sepharose column illustrated the hirudinS protein band with size about ∼10.8. Concentration of produced hirudin within its solution reached to 1.75 ng, thrombin titration method showed that the hirudin protein required 300 µl from thrombin to clot, also, APPT test showed that hirudin elongated clotting time to 7 min in comparison with 6min for aspirin and the statistical analysis results for APPT test illustrated that there was no significant difference between hirudin and aspirin. Conclusion: This study approved that overlapping extension PCR is a good strategy for building hirudin gene and it's successfully expressed in BL21(DE3) strain.

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“…Sedangkan pada seleksi koloni biru/putih, hidrolisis X-gal oleh -galactosidase akan menyebabkan warna biru pada koloni dan mengindikasikan bahwa koloni tersebut mengandung vektor tanpa DNA target. Sebaliknya koloni putih mengindikasikan bahwa koloni mengandung insersi DNA target (Padmanabhan et al, 2011). Selanjutnya dilakukan konfirmasi dengan PCR koloni serta analisis menggunakan enzim restriksi EcoR1.…”

Section: Uji Sensitivitas Primerunclassified

DETEKSI DINI Enterocytozoon hepatopenaei (EHP) PADA UDANG VANAME (Litopenaeus vannamei) MENGGUNAKAN METODE PCR (POLYMERASE CHAIN REACTION)

Faisal

Pancoro

2018

Jurnal Riset Akuakultur

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ABSTRAKSejak akhir tahun 2014, wabah kotoran putih atau yang sering disebut juga WFD (White Feces Disease), merupakan salah satu masalah yang sering terjadi pada petambak udang di Indonesia. Wabah ini diketahui disebabkan oleh Enterocytozoon hepatopenaei (EHP) dan telah mengakibatkan retardasi pertumbuhan hingga kematian pada udang. Hingga saat ini, penyakit WFD dapat dideteksi dengan cara uji histologi, hibridisasi in situ, dan PCR. Penelitian ini bertujuan untuk mendapatkan metode deteksi dini penyakit EHP pada udang vaname dengan metode PCR melalui perancangan primer yang spesifik dan sensitif. Pada penelitian ini dilakukan isolasi EHP pada udang vaname yang terinfeksi, kemudian dideteksi dengan metode PCR yang mentarget SWP (spore wall protein) dari EHP serta pengujian spesifitas dan sensitivitasnya. Hasil yang diperoleh menunjukkan bahwa EHP dapat diisolasi dari udang yang terinfeksi dan dapat didesain dua pasang primer yaitu SWP-EHP1 dan SWP-EHP3 yang mentarget spore wall protein EHP. Kedua primer ini dapat digunakan untuk deteksi EHP menggunakan PCR, dengan produk PCR pada primer SWP-EHP1 yaitu 398 bp dan primer SWP-EHP3 sebesar 415 bp, serta nilai suhu annealing optimal pada 48 o C.Hasil pengujian sensitivitas primer, diketahui bahwa primer SWP-EHP1 dapat mendeteksi EHP hingga jumlah DNA target sebanyak 7,74 x 10 2 kopi sedangkan primer SWP-EHP3 dapat mendeteksi hingga 16,2 x 10 2 kopi. KATA KUNCI: udang vaname; WFD; Enterocytozoon hepatopenaei; PCR ABSTRACT: Detection of EHP (Enterocytozoon hepatopanaei) from whiteleg shrimp (Litopenaeus vannamei) by Polymerase Chain Reaction (PCR) method. By: Annisa Fitriah Faisal and Adi PancoroSince 2014, white feces disease (WFD) is one of the emerging problems for whiteleg shrimp farming industries in Indonesia. This outbreak is known to be caused by Enterocytozoon hepatopenaei (EHP) infection to shrimp. EHP infection resulted in growth retardation to a mass mortality in shrimp. To date, WFD can be detected by histology, in situ hybridization and PCR. This study aimed to obtain an early detection method of EHP on whiteleg shrimp by PCR method through specific and sensitive primers design. In this study, we isolated the DNA of EHP from infected whiteleg shrimp, then detected by PCR method which targeted spore wall protein (SWP) from EHP as well as sensitivity and specificity testing. As a result, EHP can be isolated from infected shrimp and can be designed 2 pairs of primers (SWP-EHP1 and SWP-EHP3) targeting spore wall protein of EHP. These primers could be used for EHP detection using PCR, with PCR products from primers SWP-EHP1 was 398 bp and from SWP-EHP3 primers was 415 bp, with an optimum annealing temperature of 48 o C. Primers sensitivity test results revealed that primers SWP-EHP1 could detect EHP to 7.74 x 10 2 copies while the primers SWP-EHP3 could detect up to 16.2 x 10 2 copies.

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Screening of Bacterial Recombinants: Strategies and Preventing False Positives

Cited by 14 publications

References 58 publications

Big trouble with little inserts: boundaries in metagenomic screenings usinglacZα based vectors

Big trouble with little inserts: boundaries in metagenomic screenings usinglacZα based vectors

Production and purification of constructed recombinant hirudin in BL21(DE3) strain

DETEKSI DINI Enterocytozoon hepatopenaei (EHP) PADA UDANG VANAME (Litopenaeus vannamei) MENGGUNAKAN METODE PCR (POLYMERASE CHAIN REACTION)

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