Engineering Of Bacteria Research Articles

Folates and Folic Acid: From Fundamental Research Toward Sustainable Health

Folates are of paramount importance in one-carbon metabolism of most organisms. Plants and microorganisms are able to synthesize folates de novo, making them the main dietary source for humans and animals, which are dependent on food or feed supplies for folates. Folate deficiency is an increasing problem in the developing, as well as in the developed regions of the world, affecting millions of people. Different strategies, such as food fortification and folic acid supplementation, remain far from accessible for the poor rural populations in developing countries. Increasing knowledge concerning folate biosynthesis, transport and catabolism does not only deepen our insight on the regulation of folate metabolism but also provides the keys towards folate enhancement through metabolic engineering in bacteria, as well as in plants. Recently, promising results were obtained using such an approach, but further fundamental research is a prerequisite to develop a practicable solution to fight folate deficiency. In parallel, progress in the development and improvement of folate analysis has been made. Here, we provide the state-of-the-art of folate biosynthesis, catabolism, and salvage. Finally, we report on progress in folate biofortification and discuss the agroeconomical aspect of biofortified crop plants.

DNA signatures for detecting genetic engineering in bacteria

New computational tools were used to find a robust set of DNA oligomers that can distinguish artificial vector sequences from all available background viral and bacterial genomes.

Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

AbstractThe bacterial chromosome and plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage‐encoded recombination functions efficiently to recombine sequences with homologies as short as 35 to 40 bases. This recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. Support protocols describe a two‐step method of making genetic alterations without leaving any unwanted changes, and a method for retrieving a genetic marker (cloning) from the E. coli chromosome or a co‐electroporated DNA fragment and moving it onto a plasmid. A method is also given to screen for unselected mutations. Additional protocols describe removal of defective prophage, methods for recombineering.

Meet the Guest Editor

Dr. Edgardo T. Farinas did his undergraduate work at Loyola University of Chicago (B.S. in 1990) and pursued graduate studies in bioinorganic chemistry at the University of California at Santa Cruz (Ph.D. in 1997) under Pradip Mascharak. After postdoctoral studies with Lynne Regan at Yale University, Frances Arnold at the California Institute of Technology, and Brent Iverson and George Georgiou at University of Texas at Austin, he joined the New Jersey Institute of Technology faculty as an Assistant Professor of Chemistry. His research has focused on the interface between chemistry, biology, and engineering. His current research interests are in engineering proteins using directed evolution and rational approaches. The goals include developing high-throughput screening technologies to assay mutant enzyme libraries to discovery novel biocatalyst, combining rational and directed evolution approaches to create de novo enzymes, metabolic pathway engineering in bacteria, novel protein display technologies, and incorporation of non-natural amino acids in proteins. SELECTED PUBLICATIONS [1] Alcalde, M.; Farinas, E.T.; Arnold, F.H. J. Biomolecular Screen., 2004, 9, 141-146. [2] Farinas, E.T.; Alcalde, M.; Arnold, F.H. Tetrahedron, 2003, 60, 525-528. [3] Glieder, A.; Farinas, E.T.; Arnold, F.H. Nat. Biotechnol., 2002, 20, 1135-1139. [4] Farinas, E.T.; Schwaneberg, U.; Glieder, A.; Arnold, F.H. Advanced Synthesis and Catalysis, 2001, 343, 601-606. [5] Schwaneberg, U.; Otey, C.; Cirino, P.C.; Farinas, E.T.; Arnold, F.H. J. Biomolecular Screen., 2001, 6, 111-118. [6] Farinas, E.T.; Regan, L. Protein Science, 1998, 7, 1939-1946. [7] Farinas, E.T.; Nguyen, C.; Mascharak, P.K. Inorganica Chimica Acta, 1997, 263, 17-21. [8] Farinas, E.T.; Tan, J.D.; Mascharak, P.K. Inorg. Chem., 1996, 35, 2637. [9] Guajardo, R.J.; Chavez, F.; Farinas, E.T.; Mascharak, P.K. J. Am. Chem. Soc., 1995, 117, 3883. [10] Farinas, E.T.; Baidya, N.; Mascharak, P.K. Inorg. Chem., 1994, 33, 5970. [11] Tan, J.D.; Farinas, E.T.; David, S.S.; Mascharak, P.K. Inorg. Chem., 1994, 33, 4295. [12] Farinas, E.T.; Tan, J.D.; Baidya, N.; Mascharak, P.K. J. Am. Chem. Soc., 1993, 115, 2996.

Binding of S-layer homology modules from Clostridium thermocellum SdbA to peptidoglycans

S-layer homology (SLH) module polypeptides were derived from Clostridium josui xylanase Xyn10A, Clostridium stercorarium xylanase Xyn10B, and Clostridium thermocellum scafoldin dockerin binding protein SdbA as rXyn10A-SLH, rXyn10B-SLH, and rSdbA-SLH, respectively. Their binding specificities were investigated using various cell wall preparations. rXyn10A-SLH and rXyn10B-SLH bound to native peptidoglycan-containing sacculi consisting of peptidoglycan and secondary cell wall polymers (SCWP) prepared from these bacteria but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWP, suggesting that SCWP are responsible for binding with SLH modules. In contrast, rSdbA-SLH interacted with HF-EPCS, suggesting that this polypeptide had an affinity for peptidoglycans but not for SCWP. The affinity of rSdbA-SLH for peptidoglycans was confirmed by a binding assay using a peptidoglycan fraction prepared from Escherichia coli cells. The SLH modules of SdbA must be useful for cell surface engineering in bacteria that do not contain SCWP.

Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

AbstractThe bacterial chromosome and plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage‐encoded recombination functions efficiently recombine sequences with homologies as short as 35 to 40 bases. This recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with double‐stranded DNA (dsDNA) or single‐stranded DNA (ssDNA). Support protocols describe a two‐step method of making genetic alterations without leaving any unwanted changes, and a method for retrieving a genetic marker (cloning) from the E. coli chromosome or a coelectroporated DNA fragment and moving it onto a plasmid. A method is also given to screen for unselected mutations. Additional protocols describe removal of defective prophage, methods for recombineering.

Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

AbstractThe bacterial chromosome and plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage‐encoded recombination functions efficiently recombine sequences with homologies as short as 35 to 40 bases. This recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with double‐stranded DNA (dsDNA) or single‐stranded DNA (ssDNA). Support protocols describe a two‐step method of making genetic alterations without leaving any unwanted changes, and a method for retrieving a genetic marker (cloning) from the E. coli chromosome or a coelectroporated DNA fragment and moving it onto a plasmid. A method is also given to screen for unselected mutations. Additional protocols describe removal of defective prophage, methods for recombineering.

Detection of illegal growth promoters in biological samples using receptor binding assays

In the European Union (EU), the use of growth-promoting substances in meat production is banned. The control of growth promoters, especially steroid hormones, is presently based on expensive and time-consuming chromatographic methods of analysis or, sometimes, for screening purposes, on radio- or enzyme-immunoassays, all of which are often too specific to allow effective multi-analyte control. In order to develop rapid and inexpensive multi-analyte detection tests, we proposed the use of hormonal receptors as detection tools. The system described here (radio-receptor assays) is based on a direct binding assay of steroid hormones to their respective receptors. Human receptors to estrogens (hERα), androgens (hAR), progestagens (hPR) and glucocorticoids (hGR) have been produced by genetic engineering in bacteria or in eucaryotic cells. Binding analyses revealed that the obtained receptor proteins retained a high affinity for their corresponding native ligand. In addition, competition studies confirmed that each of the four receptors displays a specificity profile for a series of analogs in agreement with the literature. Finally, the stability of these recombinant receptors is sufficient to allow their use in test kits.

Genetic engineering of bacteria and viruses

Genetic engineering holds out great promise for the development of improved poultry vaccines. Engineered microbes containing foreign genes from several pathogens provide the basis for multivalent vaccines. The vaccine vector should be non-pathogenic and capable of incorporating and expressing foreign genes. Subunit proteins expressed by non-avian vectors, such as baculoviruses, can be used to evaluate the nature of their antigenic and protective properties and could find use as subunit or virus-like particle based vaccines. Live recombinant viruses can also be used as vaccines. While RNA viruses such as picornaviruses have been used as vectors, DNA viruses are more easily engineered. Avian poxviruses expressing the Newcastle disease virus (NDV) haemagglutin neuraminidase (HN) and fusion (F) proteins protect against Newcastle disease. Because of their ease of administration through the feed or water, their ability to establish mucosal immunity, and recent advances in genome manipulation, avian adenoviruses are now being developed as recombinant avian vaccines. A recombinant fowl adenovirus expresses a green fluorescent protein reporter gene cloned into a non essential TR-2 region of viral DNA and is also immunogenic. Current efforts are directed at incorporating genes, such as HN from NDV, from avian pathogens into this vector. Genetic engineering can also improve diagnostics. For example, recombinant HN can be used in enzyme-linked immunosorbent assay (ELISA) based NDV diagnostic kits. Baculovirus expressed NDV nucleoprotein (NP) used in a differential ELISA can differentiate between birds immunised with a fowl pox virus expressing NP and NDV infected birds. Polymerase chain reaction (PCR) also has an impact on avian health. PCR can be used in diagnostics and, because of its extreme specificity, in epidemiological studies. Rapid and accurate identification and emerging or re-emerging microbial diseases are some challenges that can be more readily addressed by the application of molecular approaches including genetic engineering. Similarly, the development of multivalent and multi-pathogen vaccines through genetic engineering is certainly a realistic objective. Such vaccines would provide multiple protection in a cost effective manner.

98/03658 Metabolic engineering of bacteria for ethanol production

98/03658 Metabolic engineering of bacteria for ethanol production

Metabolic engineering of bacteria for ethanol production

Metabolic engineering of bacteria for ethanol production