Molecular Cloning

Molecular cloning is a process that manipulates the spider silk sequence to select for a proper sequence size and specific vector into which it can be expressed. This process is achieved using techniques such as digests, transformations, purifications, and ligations. This semester, our work has centered around the mPRI(alfalfa), pOET2(insect), and pmk(ecoli) vectors, into which we have, or will, insert 3 time, 6 time, and 9 time sequences of the spider silk amino acid chain. With each repetition of the silk sequence, its properties improve and become more like natural silk. Vector specification allows these sequences to be produced in different hosts, with each providing beneficial and detrimental circumstances to the silks eventual development and production. These processes are ongoing but we hope to produce usable protein in both the mPRI and pOET2 vectors in the coming months, allowing us to refine our production process until the best possible product is achieved

In vivo DNA assembly using common laboratory bacteria: A re-emerging tool to simplify molecular cloning

Molecular cloning is a cornerstone of biomedical, biotechnological, and synthetic biology research. As such, improved cloning methodologies can significantly advance the speed and cost of research projects. Whereas current popular cloning approaches use in vitro assembly of DNA fragments, in vivo cloning offers potential for greater simplification. It is generally assumed that bacterial in vivo cloning requires Escherichia coli strains with enhanced recombination ability; however, this is incorrect. A widely present, bacterial RecA-independent recombination pathway is re-emerging as a powerful tool for molecular cloning and DNA assembly. This poorly understood pathway offers optimal cloning properties (i.e. seamless, directional, and sequence-independent) without requiring in vitro DNA assembly or specialized bacteria, therefore vastly simplifying cloning procedures. Although the use of this pathway to perform DNA assembly was first reported over 25 years ago, it failed to gain popularity, possibly due to both technical and circumstantial reasons. Technical limitations have now been overcome, and recent reports have demonstrated its versatility for DNA manipulation. Here, we summarize the historical trajectory of this approach and collate recent reports to provide a roadmap for its optimal use. Given the simplified protocols and minimal requirements, cloning using in vivo DNA assembly in E. coli has the potential to become widely employed across the molecular biology community

QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids

Background Molecular cloning is an essential step in biological engineering. Methods involving megaprimer-based PCR of a whole plasmid are promising alternatives to the traditional restriction-ligation-based molecular cloning. Their widespread use, however, is hampered by some of their inherent characteristics, e.g., linear amplification, use of self-annealing megaprimers and difficulty with performing point insertion of DNA. These limitations result in low product yield and reduced flexibility in the design of a genetic construct. Result Here, we present a novel technique of directional cloning, which overcomes these problems yet retaining the simplicity of whole-plasmid amplification. QuickStep-Cloning utilizes asymmetric PCRs to create a megaprimer pair with 3′-overhangs, and hence, facilitates the subsequent exponential whole-plasmid amplification. QuickStep-Cloning generates nicked-circular plasmids, thereby permitting direct bacterial transformation without DNA ligation. It allows DNA fragment integration into any plasmid at any position, in an efficient, time- and cost-effective manner, without tedious intermediate DNA gel purification, modified oligonucleotides, specialty enzymes and ultra-competent cells. The method is compatible with competent E. coli cells prepared using the conventional calcium chloride method. Conclusion QuickStep-Cloning expands the versatility of megaprimer-based cloning. It is an excellent addition to the cloning toolbox, for the benefit of protein engineers, metabolic engineers and synthetic biologists

Engineering a minimal cloning vector from a pUC18 plasmid backbone with an extended multiple cloning site

Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185 bp fully functional high-copy cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described

Diversity of actinobacteria in the marshes of Ezzemoul and Djendli in northeastern Algeria

The main purpose of this research is to study the microbial diversity of actinobacteria, living in “Ezzemoul” and “Djendli” sebkhas soils. These salt lakes are situated in the east of Algeria and they are microbiologically underexploited. Such unexplored ecological niches have been considered by many authors as sources of novel actinobacteria and bioactive molecules. Actinobacteria play an important role in safeguarding the environment by improving plant growth through nitrogen fixation, biodegradation, and bioremediation. Therefore, studying the diversity and distribution of actinobacteria in such special environments is important for determining the ecological and biotechnological roles of these microorganisms. In this article, we focused on the occurrence and the diversity of actinobacteria from sebkhas using two techniques: cultural and culture-independent (molecular cloning). The latter are based on phylogenetic analysis of the 16S rDNA gene. Thus, the cultural method allowed us to obtain 62 isolates: 40 from the “Ezzemoul” site and 22 from the “Djendli” site. These isolates tolerate mainly 2, 5, and 10% sodium chloride (NaCl) and belong to the genera Nocardiopsis, Streptomyces, and Rhodococcus. Moreover, the molecular cloning gave us 39 clones. Twenty-four clone sequences from “Ezzemoul” site are affiliated to the genera Demequina, Plantactinospora, Friedmanniella, and Mycobacterium. Also, 15 clone sequences from “Djendli” site are related to the genera Marmoricola, Phytoactinopolyspora, Streptomyces, and to an unclassified actinobacterial clone. Some sequences from both sites are related to uncultured clones. In addition to the data provided by the cultural method, molecular cloning allowed us to have additional information about the unknown actinobacteria, uncultured ones as well as on the genera that exist in both sites. So, the cultural method is complementary to the culture-independent one, and their combination revealed an important diversity in targeted saline environments. Furthermore, all new isolated strains that tolerate 10% NaCl may have a very interesting biotechnological potential in the future

Ion channels: structural basis for function and disease.

Ion channels are ubiquitous proteins that mediate nervous and muscular function, rapid transmembrane signaling events, and ionic and fluid balance. The cloning of genes encoding ion channels has led to major strides in understanding the mechanistic basis for their function. These advances have shed light on the role of ion channels in normal physiology, clarified the molecular basis for an expanding number of diseases, and offered new direction to the development of rational therapeutic interventions

Engineering a minimal cloning vector from a pUC18 plasmid backbone with an extended multiple cloning site

Minimal plasmids play an essential role in many intermediate steps in molecular biology. For example, they can be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185-bp fully functional high-copy cloning plasmid with an extended multiple cloning site. We believe that this is the smallest high-copy cloning vector ever described. METHOD SUMMARY: We eliminated all superfluous sequences in a commonly used cloning vector in order to generate as small a cloning plasmid as possible by simple iterative PCR mutagenesis

Molecular cloning of the cDNA encoding pp36, a tyrosine-phosphorylated adaptor protein selectively expressed by T cells and natural killer cells.

Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cgamma-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell-specific protein with several putative Src homology 2 domain-binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cgamma-1 and Grb2. Studies with GST-Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation