The GECKIL Genetic Engineering Laboratory
The Vitreoscilla hemoglobin
(# 1 modulator in metabolic engineering)
The Vitreoscilla hemoglobin (commonly abbreviated as VHb) was first discovered by Dale A. Webster in 1986 (1). However, Dale (latter to be my co-advisor together with Benjamin C. Stark, both at IIT, Chicago) originaly discovered it in 1966 (2) and given its prevalent presence in the cytoplasm but not in the membrane fraction, he named it as "the soluble form of the cytochrome bo", the terminal oxidase of bacterial respiratory chain.
I had the privilege to be one of the firsts to study the VHb and its gene (vgb) with Dale and Ben. Ben has also been known for his discovery of "catlytic RNA" during his postdoc study at Yale University, securing the Nobel Prize in Chemistry in 1989 for his advisor Sydney Altman. Having the opportunity to work with such high-caliber scientists was the most revarding part of my life with science. Robert M. Roth, then the chairman of the Biology Department, was another mentor whom I liked how he orchestrated his teaching with an explicit Brooklyn accent. Thank to all three for believing in me, a naive Turkish native, to be their TA for their courses (Advanced Biochemistry by Dale, Molecular Cell Biology by Ben, and Microbial Genetics and Genetic Engineering by Bob).
For sometime, the hemoglobins were considered to be the proteins of only eukaryotic origin. The main reason for this view is that, most eukaryotic organisms (with the exeption of the single celled ones) are multicellular and the free diffusion of oxygen into cells deep in the thick tissues and organs would not be possible without a suitable carrier (i.e. hemoglobin). Now, we know that hemoglobins exisit virtually in all kingdoms of life. But, what role such proteins may play in free living single cells, such as bacteria, which acquire oxygen from the environment readily through passive diffusion. Although, this is a question of ongoing debate, we now also know that not only do these ubiquitous protiens transport the oxygen between tissues, the known function of hemoglobins, but also they play other roles ranging from intracellular oxygen transport to catalysis of redox reactions.
Aerobic (oxygen using) organisms started to dominate the life on earth about 3 billion years ago, as the anoxic (oxgen-free) atmosphere become more and more oxic (oxygenated) with the advent of photosynthesis about 4 billion years ago. With some minor exeptions (e.g., anerobic bacteria), today life on earth in all levels, as we know it, is oxygen dependent and it is no surprise that many genes (if not all) in all these life forms from bacteria to human are directly or inderectly regulated by oxygen. Moreover, oxygen is the final electron acceptor in the respiratory chain which is coupled to oxidative phosphorylation through which all organisms generate about 90 % of thier energy (i.e., ATP). So without this gas, i.e., oxygen, life as a whole would be succumed to death.
In multicellular organsims with bulky bodies, oxygen transfer into the cells residing far from perifery would be a problem if not solved by a specilized carrier. Because, oxyen is easly disolved in cellular and extracellular milieu where many oxygen hungry molecules or reactions can readly consume it, hindering its further transfer. Life solved this problem by creating hemoglobins and veins. The tracks, that are veins, provide blood cells (i.e. erythrocytes) which carry oxygen loaded hemoglobin to their destination all over the body (when there is no gain to oxygen, ther is pain!). With its highly efficient oxyen-carbon dioxyde affinity kinetics, hemoglobin relaeses more oxygen to regions where there is much need (low oxygen, high carbondioxide regions).
A bacterium is both an organism and also a cell. It is not in tissue form and does not have extensive intracellular structures (i.e., organeles). As for the cells in organisms, oxygen is easly diffuesed through its membrane and the final destinations are not that far when its size is considered (~1 x 2 µm). So, why invest so much energy to synthesize a 146 amino asit long hemoglobin protein (who knows how many molecules of it).
The term "metabolic engineering" was first coined by late James E. Bailey (then at Caltech) and Gregory Stephanopoulos (Massachusetts Institute of Technology (MIT)) in 1991 in the same issue of the journal Science (3,4). Prof. Bailey was also a pioneer in VHb studies (and his group was a rival of our laboratory at IIT) and Prof. Stephanopoulos is the editor-in-chief of the journal Metabolic Engineering.
Genetic Engineering of Vitreoscilla Hemoglobin Gene
Metabolic Reprogramming and Cancer
1. Wakabayashi, S.; Matsubara, H.; Webster, D. A., Primary Sequence of a Dimeric Bacterial Hemoglobin from Vitreoscilla. Nature 1986, 322, (6078), 481-483.
2. Webster, D. A.; Hackett, D. P., The purification and properties of cytochrome o from Vitreoscilla. J Biol Chem 1966, 241, 3308-3315.
3. Bailey, J. E., Toward a Science of Metabolic Engineering. Science 1991, 252, (5013), 1668-1675.
4. Stephanopoulos, G.; Vallino, J. J., Network Rigidity and Metabolic Engineering in Metabolite Overproduction. Science 1991, 252, (5013), 1675-1681.
5. Geckil, H.; Stark, B. C.; Webster, D. A., Cell growth and oxygen uptake of Escherichia coli and Pseudomonas aeruginosa are differently effected by the genetically engineered Vitreoscilla hemoglobin gene. Journal of Biotechnology 2001, 85, (1), 57-66.
6. Geckil, H.; Gencer, S.; Kahraman, H.; Erenler, S. O., Genetic engineering of Enterobacter aerogenes with theVitreoscilla hemoglobin gene: cell growth, survival, and antioxidant enzyme status under oxidative stress. Research in Microbiology 2003, 154, (6), 425-431.
7. Geckil, H.; Barak, Z.; Chipman, D. M.; Erenler, S. O.; Webster, D. A.; Stark, B. C., Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene. Bioprocess and Biosystems Engineering 2004, 26, (5), 325-330.
8. Erenler, S. O.; Gencer, S.; Geckil, H.; Stark, B. C.; Webster, D. A., Cloning and expression of the Vitreoscillahemoglobin gene in Enterobacter aerogenes: Effect on cell growth and oxygen uptake. Applied Biochemistry and Microbiology 2004, 40, (3), 241-248.
9. Geckil, H.; Gencer, S., Production of L-asparaginase in Enterobacter aerogenes expressing Vitreoscillahemoglobin for efficient oxygen uptake. Applied Microbiology and Biotechnology 2004, 63, (6), 691-697.
10. Kurt, A. G.; Aytan, E.; Ozer, U.; Ates, B.; Geckil, H., Production of L-DOPA and dopamine in recombinant bacteria bearing the Vitreoscilla hemoglobin gene. Biotechnology Journal 2009, 4, (7), 1077-88.
11. Erenler, S.; Geckil, H., Cloning, isolation and expression of L-asparaginase gene (ansB) in different gram-negative bacteria expressing Vitreoscilla hemoglobin. Current Opinon in Biotechnology 2011, 22, S118-S118.
Periodic Table of the People
ACADEMIC SUPERVISING (ongoing...)
"Any intelligent fool can make things bigger, more complex and more violent. It takes a touch of genius and a lot of courage to move in the opposite direction" - Albert Einstein
PROJECTS (as PI)
RESEARCH PROJECTS REVIEW PANELS
THE GECKIL GENETIC ENGINEERING LABORATORY is located in the west wing of the colossal E-shaped Advanced Research Center (ARC) of Inonu University. The laboratory is fully equipped to support molecular biology and genetic engineering studies. DNA amplification, gene cloning and expression, pathway and metabolic engineering are the type of rearch methodologies being carried out. Some basic tools of the trade in our lab:
Some other ARC instrumentation at our disposal:
Databases and Computational Tools
1.1.4. GenBank ftp site
1.2.1. Rfam: RNA familiy database
1.2.3. sRNA: Small RNAdatabase
1.3. Comparative & Phylogenetic
1.4. SNPs, Mutations & Variations
1.5. Microarray & Gene Regulation
1.5.2. Array Express
1.5.8. The Signaling Gateway
1.6. Proteins & Interactions
1.7. Reaction Pathways
1.7.2. KEGG ftp site
1.8. Enzyme Databases
1.9. Membrane Transporters
1.10.1. Functional Glycomics
1.11. Protein Structure
1.11.1. Protein Data Bank
1.11.3. Protein Information Resource
1.12. Systems Biology
1.12.1. BiGG Database
1.13. Synthetic Biology
1.13.1. Standard Biological Parts
1.14.1. Gene Ontology
2. Computational Tools
2.1. Genome Browser
2.2. Sequences Comparison & Alignment:
2.2.1. NCBI BLAST
2.3. Promotor & Transcription Regulation
2.3.4. E. coli DNA-Binding Site
2.4. Microarray & Gene Regulation
2.4.2. MeV: MultiExperiment Viewer
2.4.6. Agilent eArray
2.5. Membrane Protein Analysis
2.6. Proteomics / Mass Spectrometry
2.6.1. ExPASy Tools
2.7. Protein Structure Visualization
2.8.2. COBRA Toolbox
2.9.2. KEGG Tools
2.9.4. Extreme Pathway Analysis
2.10. RNA folding
2.10.3. siRNA SelectionProgram
2.11. Oligomer Microarray Design
2.11.2. OligoArray 2.0
2.11.4. OligoWiz 2.0
2.11.10. EC Oligos
2.12. Protein 2-D Structure
2.13. Protein 3-D Structure
2.13.2. CPHmodels 2.0
2.13.4. Topology of Protein Structure
2.14. Systems Biology
2.14.2. Systems Biology Workbench
2.14.5. Gene Designer
2.14.6. Systems Biology Markup Language
2.14.8. Tools at Weiznmann Institute
Fulbright Scholars Program awards 21 from Harvard with grants Harvard Gazette 2010
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