OverFlap PCR – a reliable approach for generation of plasmid DNA libraries containing random sequences without template bias.

Over the decades the improvement of naturally occurring proteins and creation of novel ones has been the primary goal for many practical biotechnology researchers and it is widely recognized that randomization of protein sequences coupled to various effect screening methodologies is one of the most powerful techniques for fast, efficient and purposeful approach for acquisition of desired improvements. Over the years considerable advancements have been made in this field, however development of PCR based or template guided methodologies has been hampered by the resulting template sequence bias. In this article we present novel whole plasmid amplification based approach, which we named OverFlap PCR, for randomization of virtually any region of the plasmid DNA, without introduction of mentioned bias.

PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries

QuickStep is a cloning method that allows seamless point integration of a DNA sequence at any position within a target plasmid using only Q5 High-Fidelity DNA Polymerase and DpnI endonuclease. This efficient and cost-effective method consists of two steps: two parallel asymmetric PCRs, followed by a megaprimer-based whole-plasmid amplification. To further simplify the workflow, enhance the efficiency, and increase the uptake of QuickStep, we replaced the asymmetric PCRs with a conventional PCR that uses phosphorothioate (PTO) oligos to generate megaprimers with 3′ overhangs. The ease and speed of PTO-QuickStep were demonstrated through (1) right-first-time cloning of a 1.8 kb gene fragment into a pET vector and (2) creating a random mutagenesis library for directed evolution. Unlike most ligation-free random mutagenesis library creation methods (e.g., megaprimer PCR of whole plasmid [MEGAWHOP]), PTO-QuickStep does not require the gene of interest to be precloned into an expression vector to prepare a random mutagenesis library. Therefore, PTO-QuickStep is a simple, reliable, and robust technique, adding to the ever-expanding molecular toolbox of synthetic biology and expediting protein engineering via directed evolution.

Kloning Gen pcbC dari Penicillium chrysogenum ke dalam Plasmid pPICZA untuk Pengembangan Produksi Penisilin G

Availability of drugs in Indonesia is still limited by the high prices of drugs due to on the imported raw materials that reaches 95%. Developing antibiotic raw materials can be achieved by increasing of penicillin G production, which is the raw material for the formation of semisynthetic penicillin derivatives through the production of 6-aminopenisillanic acid (6-APA). One of the important enzyme in the penicillin G biosynthesis is Isopenisilin N Synthase (IPNS) that encodes by pcbC gene on Penicillium chrysogenum. This study aimed to obtain a recombinant of pcbC gene fragments that is inserted into pPICZA plasmid. Amplification of pcbC gene used pcbC-F and pcbC-R primers. The pcbC gene fragment was inserted into pPICZA vector and then transformed into TOP 10 F’. The results showed that the recombinant of the pcbC gene fragment from P. chrysogenum has been obtained. Analysis of DNA sequences using the BLAST program showed that the pcbC gene fragment has high homology (99%) with the pcbC gene from P. chrysogenum Wisconsin 54-1255 and P. chrysogenum AS-P-78 which encodes IPNS Keywords: pcbC Gene, Penicillium chrysogenum, cloning, penicillin G

The E8∧E2 Gene Product of Human Papillomavirus Type 16 Represses Early Transcription and Replication but Is Dispensable for Viral Plasmid Persistence in Keratinocytes

ABSTRACT A conserved E8∧E2 spliced mRNA is detected in keratinocytes transfected with human papillomavirus type 16 (HPV-16) plasmid DNA. Expression of HPV-16 E8∧E2 (16-E8∧E2) is independent of the major early promoter, P97, and is modulated by both specific splicing events and conserved cis elements in the upstream regulatory region in a manner that differs from transcriptional regulation of other early viral genes. Mutations that disrupt the predicted 16-E8∧E2 message also increase initial HPV-16 plasmid amplification 8- to 15-fold and major early gene (P97) transcription 4- to 5-fold over those of the wild type (wt). Expressing the 16-E8∧E2 gene product from the cytomegalovirus (CMV) promoter represses HPV-16 early gene transcription from P97 in a dose-dependent manner, as detected by RNase protection assays. When expressed from the CMV promoter, 16-E8∧E2 also inhibits the amplification of an HPV-16 plasmid and a heterologous simian virus 40 (SV40) ori plasmid that contains E2 binding sites in cis. In contrast, cotransfections with HPV-16 wt genomes that express physiologic levels of 16-E8∧E2 are sufficient to repress HPV-16 plasmid amplification but are limiting and insufficient for the repression of SV40 amplification. 16-E8∧E2-dependent repression of HPV-16 E1 expression is sufficient to account for this observed inhibition of initial HPV-16 plasmid amplification. Unlike with other papillomaviruses, primary human keratinocytes immortalized by the HPV-16 E8 mutant genome contain more than eightfold-higher levels of unintegrated plasmid than the wt, demonstrating that 16-E8∧E2 limits the viral copy number but is not required for plasmid persistence and maintenance.

Polymerases Leave Fingerprints: Analysis of the Mutational Spectrum in Escherichia coli rpoB To Assess the Role of Polymerase IV in Spontaneous Mutation

ABSTRACT We compared the distribution of mutations in rpoB that lead to rifampin resistance in strains with differing levels of polymerase IV (Pol IV), including strains with deletions of the Pol IV-encoding dinB gene, strains with a chromosomal copy of dinB, strains with the F′128 plasmid, and strains with plasmid amplification of either the dinB operon (dinB-yafNOP) or the dinB gene alone. This analysis identifies several hot spots specific to Pol IV which are virtually absent from the normal spontaneous spectrum, indicating that Pol IV does not contribute significantly to mutations occurring during exponential growth in liquid culture.

Stabilization of a Full-Length Infectious cDNA Clone of Transmissible Gastroenteritis Coronavirus by Insertion of an Intron

ABSTRACT The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in Escherichia coli cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different positions allowed stable plasmid amplification for at least 200 generations. Infectious TGEV was efficiently recovered from cells transfected with the modified cDNAs.