Name: functional complementation analysis (fca): a laboratory exercise designed and implemented to supplement the teaching of biochemical pathways
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Description: Watch this Scientific Journal Video about Functional Complementation Analysis FCA: A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways at JoVE.com

AOH and MAS acknowledges the College of Science and the Thomas H. Gosnell School of Life Sciences at the Rochester Institute of Technology for support. This work was supported in part by United States National Science Foundation (NSF) award to AOH MCB-1120541.

NOTE: The authors are willing to provide bacterial strains and recombinant plasmids to facilitate the incorporation of functional complementation analysis for teaching purposes for individuals who are interested. The plasmids that were used to facilitate functional complementation experiments are listed in Table 1.
1. Construction of Plasmids for Functional Complementation
<ol>
	<li>Cloning of Diaminopimelate Aminotransferase (dapL) for Functional Complementation.
	<ol>
		<l...

<ol>
	<li>Gillum, A. M., Tsay, E. Y., Kirsch, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+the+Candida+albicans+gene+for+orotidine-5'-phosphate+decarboxylase+by+complementation+of+S.+cerevisiae+ura3+and+E.+coli+pyrF+mutations.">Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations.</a> Mol Gen Genet. 198, 179-182 (1984).</li><li>Hudson, A. O., Singh, B. K., Leustek, T., Gilvarg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+L,L-diaminopimelate+aminotransferase+defines+a+novel+variant+of+the+lysine+biosynthesis+pathway+in+plants.">An L,L-diaminopimelate aminotransferase defines a novel variant of the lysine biosynthesis pathway in plants.</a> Plant Physiol. 140, 292-301 (2006).</li><li>Jander, G., Joshi, V., Last, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspartate-derived+amino+acid+biosynthesis+in+Arabidopsis+thaliana.">Aspartate-derived amino acid biosynthesis in Arabidopsis thaliana.</a> The Arabidopsis Book. , (2009).</li><li>Prabhu, P., Hudson, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+partial+characterization+of+an+L-Tyrosine+aminotransferase+from+Arabidopsis+thaliana.">Identification and partial characterization of an L-Tyrosine aminotransferase from Arabidopsis thaliana.</a> Biochemistry Research International. , 549572 (2010).</li><li>Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., Dow, M., Verdier, V., Beer, S. V., Machado , M. A., Toth, I., Salmond, G., Foster, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top+10+pathogenic+bacteria+in+molecular+pathology.">Top 10 pathogenic bacteria in molecular pathology.</a> Molecular Plant Pathology. 13, 614-629 (2012).</li><li>McGroty, S. E., Pattaniyil, D. T., Patin, D., Blanot, D., Ravichandran, A. C., Suzuki, H., Dobson, R. C., Savka, M. A., Hudson, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+Characterization+of+UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:+meso-2,6-diaminopimelate+ligase+(MurE)+from+Verrucomicrobium+spinosum+DSM+4136T.">Biochemical Characterization of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase (MurE) from Verrucomicrobium spinosum DSM 4136T.</a> PLoS ONE. 8 (6), e66458 (2013).</li><li>Hutton, C. A., Perugini, M. A., Gerrard, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+lysine+biosynthesis:+an+evolving+antibiotic+strategy.">Inhibition of lysine biosynthesis: an evolving antibiotic strategy.</a> Mol Biosyst. 3, 458-465 (2007).</li><li>Potrykus, K., Cashel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(p)ppGpp:+still+magical.">(p)ppGpp: still magical.</a> Annu Rev Microbiol. 62, 35-51 (2008).</li><li>Dalebroux, Z. D., Svensson, S. L., Gaynor, E. C., Swanson, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppGpp+Conjures+Bacterial+Virulence.">ppGpp Conjures Bacterial Virulence.</a> Microbiology and Molecular Biology Reviews. 74, 171-199 (2010).</li><li>Cashel, M., Gentry, D. J., Hernandez, V. J., Vinella, D., Neidhardt, F. C., Curtiss, R., Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., Umbarger, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stringent+response.">The stringent response.</a> Escherichia coli and Salmonella typhimurium: cellular and molecular biology. , 1458-1496 (1996).</li><li>Chatterji, D., Ojha, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+the+stringent+response,+ppGpp+and+starvation+signaling.">Revisiting the stringent response, ppGpp and starvation signaling.</a> Curr. Opin. Microbiol. 4, 160-165 (2001).</li><li>Jain, V., Kumar, M., Chatterji, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppGpp:+stringent+response+and+survival.">ppGpp: stringent response and survival.</a> J. Microbiol. 44, 1-10 (2006).</li><li>Kramer, G. F., Baker, J. C., Ames, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-UV+stress+in+Salmonella+typhimurium:+4-thiouridine+in+tRNA,+ppGpp,+and+pppGpp+as+components+of+an+adaptive+response.">Near-UV stress in Salmonella typhimurium: 4-thiouridine in tRNA, ppGpp, and pppGpp as components of an adaptive response.</a> J. Bacteriol. 170, 2344-2351 (1988).</li><li>Braeken, K., Moris, M., Daniels, R., Vanderleyden, J., Michiels, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+horizons+for+(p)ppGpp+in+bacterial+and+plant+physiology.">New horizons for (p)ppGpp in bacterial and plant physiology.</a> Trends Microbiol. 14, 45-54 (2006).</li><li>Joseleau-Petit, D., Vinella, D., D'Ari, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+alarms+and+cell+division+in+Escherichia+coli.">Metabolic alarms and cell division in Escherichia coli.</a> J. Bacteriol. 181, 9-14 (1999).</li><li>Mittenhuber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genomics+and+evolution+of+genes+encoding+bacterial+(p)+ppGpp+synthetases/hydrolases+(the+Rel,+RelA,+and+SpoT+proteins).">Comparative genomics and evolution of genes encoding bacterial (p) ppGpp synthetases/hydrolases (the Rel, RelA, and SpoT proteins).</a> J. Mol. Microbiol. Biotechnol. 3, 585-600 (2001).</li><li>Gan, H. M., Buckley, L., Szegedi, E., Hudson, A. O., Savka, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+rsh+Gene+from+a+Novosphingobium+sp.+Necessary+for+Quorum-Sensing+Signal+Accumulation.">Identification of an rsh Gene from a Novosphingobium sp. Necessary for Quorum-Sensing Signal Accumulation.</a> J. Bacteriol. 191, 2551-2560 (2009).</li><li>Nachar, V. R., Savka, F. C., McGroty, S. E., Donovan, K. A., North, R. A., Dobson, R. C., Buckley, L. J., Hudson, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+biochemical+analysis+of+the+diaminopimelate+and+lysine+biosynthesis+pathway+in+Verrucomicrobium+spinosum:+Identification+and+partial+characterization+of+L,L-diaminopimelate+aminotransferase+and+UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-meso-diaminopimelate+ligase.">Genomic and biochemical analysis of the diaminopimelate and lysine biosynthesis pathway in Verrucomicrobium spinosum: Identification and partial characterization of L,L-diaminopimelate aminotransferase and UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-meso-diaminopimelate ligase.</a> Front. Microbiol. 3, 183 (2012).</li><li>Hudson, A. O., Gir&#242;n, I., Dobson, R. C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+and+preliminary+X-ray+diffraction+analysis+of+L,L-diaminopimelate+aminotransferase+(DapL)+from+Chlamydomonas+reinhardtii.">Crystallization and preliminary X-ray diffraction analysis of L,L-diaminopimelate aminotransferase (DapL) from Chlamydomonas reinhardtii.</a> Acta Cryst. 67, 140-143 (2011).</li><li>Dobson, R. C. J., Gir &#242;n, I., Hudson, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L,L-Diaminopimelate+Aminotransferase+from+Chlamydomonas+reinhardtii:+A+Target+for+Algaecide+Development.">L,L-Diaminopimelate Aminotransferase from Chlamydomonas reinhardtii: A Target for Algaecide Development.</a> PLoS ONE. 6 (5), e20439 (2011).</li><li>Guzman, L. M., Belin, D., Carson, M. J., Beckwith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+regulation,+modulation,+and+high-level+expression+by+vectors+containing+the+arabinose+pBAD+promoter.">Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter.</a> J. Bacteriol. 177, 4121-4130 (1995).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A laboratory manual. , (2001).</li><li>Lugtenberg, E. J., van Schijndel-van Dam, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+mutants+of+Escherichia+coli+K-12+with+low+activity+of+the+diaminopimelic+acid+adding+enzyme.">Temperature-sensitive mutants of Escherichia coli K-12 with low activity of the diaminopimelic acid adding enzyme.</a> J. Bacteriol. 110, 41-46 (1972).</li><li>Mavrides, C., Orr, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispecific+aspartate+and+aromatic+amino+acid+aminotransferases+in+Escherichia+coli.">Multispecific aspartate and aromatic amino acid aminotransferases in Escherichia coli.</a> J. Biol Chem. 250, 4128-4133 (1975).</li><li>Gelfand, D. H., Steinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+mutants+deficient+in+the+aspartate+and+aromatic+amino+acid+aminotransferases.">Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases.</a> J. Bacteriol. 130, 429-440 (1977).</li><li>Whitaker, R. J., Gaines, C. G., Jensen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multispecific+quintet+of+aromatic+aminotransferases+that+overlap+different+biochemical+pathways+in+Pseudomonas+aeruginosa.">A multispecific quintet of aromatic aminotransferases that overlap different biochemical pathways in Pseudomonas aeruginosa.</a> J. Biol Chem. 257, 13550-13556 (1982).</li><li>Raskin, D. M., Judson, N., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+stringent+response+in+the+essential+function+if+the+conserved+bacterial+G+protein+CgtA+in+Vibrio+cholera.">Regulation of the stringent response in the essential function if the conserved bacterial G protein CgtA in Vibrio cholera.</a> Proc Natl Acad Sci, USA. 104, 4636-4641 (2007).</li><li>Ditta, G., Stanfield, S., Corbin, D., Helinski, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad+host+range+DNA+cloning+system+for+Gram-negative+bacteria:+construction+of+a+gene+bank+of+Rhizobium+meliloti.">Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti.</a> Proc Natl Acad Sci, USA. 77, 7347-7351 (1980).</li><li>Watanabe, A., Suzuki, M., Ujiie, S., Gomi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+enzymatic+characterization+of+a+novel+&#946;-1,6-glucosidase+from+Aspergillus+oryzae.">Purification and enzymatic characterization of a novel &#946;-1,6-glucosidase from Aspergillus oryzae.</a> J Biosci Bioeng. , (2015).</li><li>Song, J. T., Lu, H., McDowell, J. M., Greenberg, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+key+role+for+ALD1+in+activation+of+local+and+systemic+defenses+in+Arabidopsis.">A key role for ALD1 in activation of local and systemic defenses in Arabidopsis.</a> Plant J. 40, 200-212 (2004).</li><li>Rost, B., Liu, J., Nair, R., Wrzeszczynski, K. P., Ofran, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+prediction+of+protein+function.">Automatic prediction of protein function.</a> Celuular and Molecular Life Sciences. 60, 2637-2650 (2003).</li><li>Norris, S. R., Shen, X., Penna, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementation+of+the+Arabidopsis+pds1+mutation+with+the+gene+encoding+p-Hyrdoxyphenylpyruvate+dioxygenase.">Complementation of the Arabidopsis pds1 mutation with the gene encoding p-Hyrdoxyphenylpyruvate dioxygenase.</a> Plant Physiol. 117, 1317-1323 (1998).</li><li>Mohler, W. A., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+and+cell+fusion+analyzed+by+lacZ+complementation+in+mammalian+cells.">Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells.</a> Proc Natl Acad Sci, USA. 93, 12423-12427 (1996).</li></ol>

Many of the courses that are integral to the Biotechnology and Molecular Bioscience curriculum at the Rochester Institute of Technology have a laboratory component in addition to the lecture portion of the course. The curriculum for the academic year 2014-2015 contains a total of 48 courses, 29 of which contain a laboratory component which represent approximately 60%. One such course is Fundamentals of Plant Biochemistry and Pathology (FPBP), a blended lecture/ laboratory course and Bioseparations: Principles and Practic...

Functional complementation in the context of elucidating the function(s)/role(s) of a gene is defined as the ability of a particular homologous or orthologous gene to restore a particular mutant with an observable phenotype to the wild-type state when the homologous or orthologous gene is introduced in cis or trans into the mutant background. This technique has been widely used to isolate and identify the function(s)/role(s) of many genes. One particular example is the isolation and identification of orotidine-5-phosphate decarboxylase from Candida albicans using the ura3 mutant of S. cerevisiae and the pyrF mutant of E. coli.1 The authors have used this technique to elucidate the function of genes that are involved in the metabolism of amino acids, peptidoglycan and the stringent response in their research programs and have incorporated this technique into their teaching programs in the Biotechnology and Molecular Bioscience (BMB) program at the Rochester Institute of Technology (RIT).
The authors teach Fundamentals of Plant Biochemistry/Pathology (FPBP) (Hudson) and Bioseparations: Principles and Practices (BPP) (Hudson/Savka), two upper division elective laboratory based courses in the BMB Program at the RIT. Since some of the topics that are discussed in the courses are affiliated with their research interests, the authors have incorporated many of the techniques and experimental tools that are used in their respective research programs into these two laboratory-based courses. One such example is functional complementation as a laboratory exercise to reinforce the lecture materials pertaining to amino acid metabolism from plants, peptidoglycan and the stringent response metabolism from bacteria.
Three of the amino acid pathways from plants that are discussed in the FPBB course are that of lysine (lys), tyrosine (tyr) and phenylalanine (phe). The lys pathway is highlighted in the course because of the importance of the amino acid as an essential amino acid for all animals particularly humans since animals lack the genetic machinery to synthesize lys de novo. In addition, it was recently discovered that plants employ a pathway for the synthesis of lys that is significantly different from that of bacteria. This discovery was partially facilitated by functional complementation of the E. coli diaminopimelate (dap) mutants using a gene that encodes the enzyme L,L-diaminopimelate aminotransferase (DapL) from the model plant Arabidopsis thaliana.2 The variant pathways for the synthesis of lys through the intermediate diaminopimelate are shown in Figure 1. In addition, the synthesis of lys facilitates through the aspartate derived family of amino acids which is highly regulated.3 In addition to their importance in protein synthesis, the pathways for tyr and phe are highlighted given their importance in serving as precursor compounds for the anabolism of phenylpropanoid compounds involved the synthesis of plant defense compounds such as: alkaloids, lignins, flavonoids, isoflavonoids, hydroxycinnamic acid among others.4 The tyr and phe pathways are also highlighted to show the difference between the plant and bacterial anabolic pathways. In bacteria, the enzyme tyrosine aminotransferase (TyrB) is involved in the anabolism of both amino acids, whereas in plants, the enzyme is primarily involved in the catabolism of tyr and phe and is not involved in the anabolism of these amino acids. (Figure 2).4
The differences between Gram positive and Gram negative bacteria regarding the structure of peptidoglycan (PG) are highlighted in the FPBP course. The PG of Gram negative bacteria is of interest regarding plant pathology based on to the fact that most plant pathogens are Gram negative. A recent review regarding the top 10 bacterial phyto-pathogens revealed that all are Gram negative. The bacteria were from the genera: Pseudomonas, Ralstonia, Agrobacterium, Xanthomonas, Erwinia, Xylella, Dickeya and Pectobacterium.5 One of the chemical differences when comparing the PG stem of Gram negative and Gram positive bacteria is the difference between the cross-linking amino acids of both types. The initial step for that different cross-linking of PG occurs in the cytoplasmic step of PG anabolism and is facilitated by the enzyme UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-meso-2,6-diaminopimelate ligase (MurE) (Figure 3A). MurE catalyzes the addition of a particular diamine compound at third position of the peptide stem.6 In most Gram negative bacteria, the penultimate lys precursor, meso-diaminopimelate (m-DAP) serves as the cross-linking amino acid and lys serves the same role in the PG of most Gram positive bacteria (Figure 3B).7 This is due to the fact that both m-DAP and lys possess two amine groups and are capable of forming two peptide bonds for peptide stem cross-linking.
In the Bioseparations: Principles and Practices (BPP) course, the differences between open and closed systems for the cultivation of bacteria and how nutrient levels will change significantly in both systems due to environmental changes are discussed. These events are linked to regulatory changes called a &#34;shift down&#34; or &#34;shift up&#34; triggered by starvation or an adequate supply of amino acids or energy. The &#34;shift down&#34; response can occur when a bacterial culture is transferred from a rich and complex medium to a chemically defined medium with a single carbon source. This change in environment leads to the rapid cessation of tRNA and rRNA synthesis. This cessation results in the lack of ribosomes, protein and DNA synthesis even though the biosynthesis of amino acids are upregulated.
Following the &#34;shift down&#34; response, the existing ribosomes are used to produce new enzymes to synthesize the amino acids no longer available in the medium or environment. After a period of time, rRNA synthesis and new ribosomes are assembled and the population of bacterial cells begins to grow although at a reduced rate. The course of events is termed the &#34;stringent response&#34; or &#34;stringent control&#34; and is an example of global cellular regulation and can be thought of as a mechanism for adjusting the cell&#39;s biosynthetic machinery to compensate for the availability of the required substrates and energy needs.8 The stringent response thus enables bacteria to respond rapidly to fluxes of nutrients in the environment and contributes and enhances the ability of bacteria to compete in environments that can change rapidly with regards to nutrient and or substrate availability.8-9 
The stringent response has an integral role in gene expression when the availability of amino acids, carbon, nitrogen, phosphate, and fatty acids are limited.8,10-14 This stringent response is coordinated by two nucleotides, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) commonly referred together as the alarmone (p)ppGpp. For example, when amino acids are limited-which can lead to a bottleneck in protein synthesis-the alarmone, guanosine 3,5-(bis) pyrophosphate (ppGpp), derived from anabolism of guanosine 3-diphosphate 5-triphosphate (pppGpp) accumulates in the cell. The change in (p)ppGpp level is involved in expression of genes that regulate the response to overcome the lack of substrates in the environment that are directly involved in cell growth and development. Two of the genes that are involved in this process are called relA and spoT. RelA is a ribosome-associated (p)ppGpp synthetase that is involved in the response to the accumulation of uncharged tRNAs that is the result of amino acid limitation. SpoT functions as a bifunctional (p)ppGpp synthetase and hydrolase. The synthetase activity of SpoT is involved in the response to the lack of carbon and fatty acid starvation.8 The RelA/SpoT homologs are widespread in plants and bacteria and are referred to as Rsh for RelA/SpoT homologs.8,10-12,16 A recent manuscript showed that there is a specific Rsh protein involved in the synthesis of these alarmones from the bacterium Novosphingobium sp Rr 2-17.17
Here we present four biochemical pathways tethered to the functional complementation assays. The complementation assays outlined in this manuscript provides an avenue to explore employing this in vivo assay as a means of identifying and or characterizing enzymes that are predicted to have unknown/putative function(s) or as teaching tools to supplement the teaching of biochemical pathways.

<table border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:223px;">E. coli mutants</td>
			<td style="width:372px;">Hudson/Savka lab or CGSC (http://cgsc.biology.yale.edu/)</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electroporator</td>
			<td>Biorad-USA</td>
			<td>1652100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electroporation Cuvettes</td>
			<td>Biorad-USA</td>
			<td>1652082</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Temperature controlled incubator</td>
			<td>Generic</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge</td>
			<td>Generic</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luria Agar</td>
			<td>Thermofisher Scientific</td>
			<td>22700025</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luria Broth</td>
			<td>Thermofisher Scientific</td>
			<td>12795084</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M9 Medium</td>
			<td>Sigma-Aldrich</td>
			<td>63011</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potato Dextrose Medium</td>
			<td>Fisher Scientfic&#160;</td>
			<td>DF0013-15-8</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>K1377</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diaminopimelate</td>
			<td>Sigma-Aldrich</td>
			<td>92591</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thymine</td>
			<td>Sigma-Aldrich</td>
			<td>T0376</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tyrosine</td>
			<td>Sigma-Aldrich</td>
			<td>T3754</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phenlylalanine</td>
			<td>Sigma-Aldrich</td>
			<td>P2126</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aspartate</td>
			<td>Sigma-Aldrich</td>
			<td>A9256</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Valine</td>
			<td>Sigma-Aldrich</td>
			<td>V0500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leucine</td>
			<td>Sigma-Aldrich</td>
			<td>L8000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoleucine</td>
			<td>Sigma-Aldrich</td>
			<td>I2752</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Uracil</td>
			<td>Sigma-Aldrich</td>
			<td>U0750</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gylcerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Arabinose</td>
			<td>Sigma-Aldrich</td>
			<td>A3256</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetracyline</td>
			<td>Sigma-Aldrich</td>
			<td>87128</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taq DNA polymerase</td>
			<td>Thermofisher Scientific</td>
			<td>10342-020</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platinum pfx DNA polymerase</td>
			<td>Thermofisher Scientific</td>
			<td>11708-013</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T4 DNA ligase</td>
			<td>Thermofisher Scientific</td>
			<td>15224-041</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E coli Dh5-alpha</td>
			<td>Thermofisher Scientific</td>
			<td align="right">18258012</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E coli Top10</td>
			<td>Thermofisher Scientific</td>
			<td>C4040-03</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pET100D/topo vector</td>
			<td>Thermofisher Scientific</td>
			<td>K100-01</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pCR2.1 Vector</td>
			<td>Thermofisher Scientific</td>
			<td>K2030-01</td>
		</tr>
	</tbody>
</table>

functional complementation analysis (fca): a laboratory exercise designed and implemented to supplement the teaching of biochemical pathways

Functional complementation assay (FCA) is an in vivo assay that is widely used to elucidate the function/role of genes/enzymes. This technique is very common in biochemistry, genetics and many other disciplines. A comprehensive overview of the technique to supplement the teaching of biochemical pathways pertaining to amino acids, peptidoglycan and the bacterial stringent response is reported in this manuscript. Two cDNAs from the model plant organism Arabidopsis thaliana that are involved in the metabolism of lysine (L,L-diaminopimelate aminotransferase (dapL) and tyrosine aminotransferase (tyrB) involved in the metabolism of tyrosine and phenylalanine are highlighted. In addition, the bacterial peptidoglycan anabolic pathway is highlighted through the analysis of the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-meso-2,6-diaminopimelate ligase (murE) gene from the bacterium Verrucomicrobium spinosum involved in the cross-linking of peptidoglycan. The bacterial stringent response is also reported through the analysis of the rsh (relA/spoT homolog) bifunctional gene responsible for a hyper-mucoid phenotype in the bacterium Novosphingobium sp. Four examples of FCA are presented. The video will focus on three of them, namely lysine, peptidoglycan and the stringent response.

The bacterial strains that are employed in the various functional complementation analyses are listed in Table 2.
Functional complementation analysis: L,L-diaminopimelate aminotransferase (dapL)
The E. coli double mutant AOH1 (&#916;dapD::Kan2, dapE6) harbors a mutation in the dapE gene and a complete deletion of the dapD gen...

Watch this Scientific Journal Video about Functional Complementation Analysis FCA: A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways at JoVE.com

Functional Complementation Analysis FCA: A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

The validation of enzymatic activities involved in biochemical pathways can be elucidated using functional complementation analysis (FCA). Described in this manuscript is the FCA assay demonstrating the enzymatic activity of enzymes involved in the metabolism of amino acids, bacterial stringent response and bacterial peptidoglycan biosynthesis.

Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

Functional complementation assay (FCA) is an in vivo assay that is widely used to elucidate the function/role of genes/enzymes. This technique is very ...

The overall goal of the Functional Complementation Essay is to elucidate the activity of an enzyme by introducing a functional copy of the corresponding gene into a mutant cell to see if it restores a wild type phenotype in the mutant background. This method can help answer key questions in a variety of fields, such as biochemistry, microbiology, genetics, plant biology, evolution, and others. The main advantage of this technique is that it assesses the function, activity, and/or role of an enzyme, under in vivo conditions, which are normally physiological in nature, as opposed to in vitro conditions, which are normally artificial in nature.The implications of this technique, extend towards identification, and/or the elucidation of the function of uncharacterized enzymes, and those that still have putative or predicted functions. Demonstrating this procedure of functional complementation is biotechnology and molecular biosciences major, Taylor Harkness, a student in my laboratory. After the cloning the DapL, TarB, MurE, and RSH genes according to the text protocol, inoculate 50 milliliters of liquid medium with a single colony of mutant bacterial cells to be transformed with a gene of interest and grow overnight.On day two, use the 50 milliliter overnight culture to inoculate 1 liter of the appropriate medium and grow at 30 degrees Celsius to lock-phase, or to an OD600 of 0.4 to 0.6. Harvest the cells by centrifugation at 5, 000 times G and four degrees Celsius for 15 minutes. Then decant the supernatant and use 500 milliliters of sterile, ice cold, distilled water to resuspend the pellet.Centrifuge the cells again, and after decanting the supernatant, use 250 milliliters of sterile, ice cold 10%glycerol to resuspend the pellet. After spinning the cells a final time, use two milliliters of 10%glycerol to resuspend the cells. Transfer 50 microliter aliquots into microcentrifuge tubes and immediately freeze by placing the tubes in a dry ice ethanol bath.Store the cells at minus 80 degrees Celsius for electroporation. To carry out electroporation, add 1 microliter of recombinant plasmid to a 50 microliter aliquot of competent cells and place on ice for five minutes. Transfer the cells to an electroporation cuvette and set the electroporation apparatus to the following settings:25 degrees Fahrenheit capacitance, 2.5 kilovolts, and 200 ohm resistance.Then with the cuvette in the device, deliver a pulse. Add 1 milliliter of recovery medium to the cuvette and transfer the cells to a 15 milliliter conical tube. Allow the cells to recover by incubating the culture in a shaking incubator with gentle rotation for 60 minutes at the appropriate temperature required by the mutant.To select for transformants, pipette 100 microliters of the culture onto medium with agar plates and use a sterile spreader to spread the solution. Then, incubate overnight at the appropriate temperature. Refer to the text protocol for further details.To perform complementation analysis, transform AOH1 with the empty vector pBAD33 or with dapL expression vectors using electroporation, as just demonstrated. Plate the transformation mixture on LB agar medium, supplemented with 50 micrograms per milliliter of Dap, 34 micrograms per milliliter of chloramphenicol, and 50 micrograms per milliliter of kanamycin. Incubate the plates at 30 degrees Celsius for 24 hours before observing the results.With the empty vector pBAD33 or with the murE expressing vector, transform the TKL-11 mutant using electroporation as demonstrated earlier in this video. Plate the transformants on LB agar, supplemented with 50 micrograms per milliliter of thiamine, and 34 micrograms per milliliter of chloramphenicol, and incubate the plates at 30 degrees Celsius for 24 hours before checking for transformants. Test for complementation by streaking or plating colonies from both the control and experimental transformations onto two plates of LB medium, plus 0.2%arabinose, and 50 micrograms per milliliter of thiamine.Incubate one plate at 30 degrees Celsius and the other at 42 degrees Celsius for 24 hours before assessing the growth phenotype. Transform the mutant Hx699 strain and the wild type Rr217 strain of novosphingobium species with the pRK290 empty vector, or a vector containing the native rshNsp gene in two separate transformation events each, as demonstrated earlier in this video. Plate the transformants on potato dextrose or PD agar supplemented with 10 micrograms per milliliter of tetracycline and incubate for at least 72 hours, or until transformants appear.Then, streak the transformants in an X-pattern on fresh PD agar plates with tetracycline. After incubating at 30 degrees Celsius for at least four days, visually examine the growth phenotype of both plates. The E.coli double mutant AOH1 harbors a mutation in the DapE gene, and a deletion of the DapD gene, and won't grow unless Dap is provided.As seen here, the AOH1 mutant expressing DapLs is able to grow on LLDap-free medium by converting THDP to LLDap in the Dap lyse pathway. The MurE enzyme facilitates the addition of m-DAP into the peptidoglycan of gram-negative bacteria. The E.coli MurE mutant, TKL-11, cannot grow at 42 degrees Celsius.In this experiment, only TKL-11 cells transformed with MurE grow at 42 degrees Celsius. A mutation in the TyrB gene prevents the synthesis of tyrosine and phenylalanine. However, as shown here, when the TyrB mutant DL39 strain expresses the A.thaliana At5g36160 gene, it grows on M9 medium, lacking tyrosine and phenylalanine, demonstrating that the enzyme can synthesize tyrosine.The Hx699 mutant in novosphingobium species harbors a non-functional RSH protein and produces hypomucoidal phenotype. However, transformation with the native RSH gene produces more soluble extracellular polysaccharides in the multicellular X-streak that is hypermucoidal. In contrast, when the Hx699 mutant is transformed with an empty vector, it produces a hypomucoidal phenotype.Once mastered, this technique can be done in approximately 24 to 48 hours. If performed properly, depending on the growth of the model organism used, excluding the cloning, competence of preparation and transformation steps. While attempting this procedure, it is important to remember the growth condition requirements of the mutants used in the functional complementation analysis.Following this procedure, other methods like traditional in-vitro axiomatic characterizations can be performed in order to answer additional questions like substrate specificity, temperature, and pH optima, and somatic velocity rates, et cetera. After its development, this technique paves the way for researchers in the field of biology and many subdivision fields, such as microbiology, to elucidate the in vivo function, or role, of enzymes from a variety of organisms. After watching this video, you should have a good understanding of how to make electrocompetent bacterial cells, how to transform bacteria using electroporation, and how to assess the function of genes/enzymes using functional complementation.Don't forget that working with pathogenic organisms can be extremely hazardous and the necessary precautions should be taken.

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