Name: 機能的相補性分析（FCA）：生化学的経路の授業を補足するように設計および実装実験演習 (動画) | JoVE Journal
Uploaded: 2016-06-24T14:00:00.000+00:00
Duration: 9 min 27 s
Description: 学ぶ機能的相補分析法による生化学的経路の酵素活性評価。変異細胞への遺伝子導入で野生型表現型を回復し、DapL・MurE・RSHなどの代謝酵素のin vivo機能を解明する教育用実験手法です。

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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>
		<li>Amplify the dapL open reading frame (ORF) from the V. spinosum and cDNAs from A. thaliana and C. reinhardtii by PCR. Use 1 cycle at 94 &#176;C for 2 min, followed by 30 cycles of 94 &#176;C for 15 sec, 60 &#176;C for 30 sec and 72 &#176;C for 2 min. Include 12 pmoles of each primer, 1 mM MgSO4, 0.5 mM each of the 4 deoxynucleotide triphosphates, and 0.5 ng of template DNA and 1 unit of Pfx DNA polymerase. 
		NOTE: The complete description pertaining to the recombinant cloning of three dapL orthologs from the plant A. thaliana (At-dapL), the bacterium Verrucomicrobium spinosum (Vs-dapL) and the alga Chlamydomonas reinhardtii (Cr-dapL) has been previously published.2,18-20 
		NOTE: The primers used for the cloning of the dapL orthologs are as follows: 
		VsdapL F- 5&#39;-CCCCGAATTCATGGCCCTCATCAACGAAAACTTCCTCAAG-3&#39; 
		VsdapL R 5&#39;- CCCCGTCGACCTACTTCAGCGCGGCGATACGGCGGCAGAC-3&#39; 
		AtdapL F- 5&#39;-GGGGCATTGGAAGGAGATATAACCATGGCAGTCAATACTTGCAAATGT-3&#39; 
		AtdapL R-5&#39;- GGGGGTCGACTCATTTGTAAAGCTGCTTGAATCTTCG-3&#39; 
		CrdapL F-5&#39;-CCCCCGAATTCATGCAGCTCAACGTGCGGTCCACCGCCAGC-3&#39; 
		CrdapL R-5&#39;- CCCCCAAGCTTCTAGTTACGCTTGCCGTAGGCCTCCTTAAA-3&#39;</li>
		<li>Clone the PCR fragments into the plasmid pET30A to produce the recombinant plasmids, pET30A::VsdapL, pET30A::AtdapL and pET30A::CrdapL using the primers, restriction enzymes and T4 DNA ligase.
		<ol>
			<li>Briefly, incubate 50 ng of insert and 20 ng of vector in 1x ligase buffer and 1 unit of T4 DNA ligase overnight at 17 &#176;C. Transform ligation into E. coli Dh5&#945; cells and screen for colonies on LB plates supplemented with 50 &#181;g/ml&#8722;1 kanamycin by incubating at 37 &#176;C for 24 hr.18-20</li>
		</ol>
		</li>
		<li>To create the plasmid for functional complementation, digest pET30A::VsdapL and pET30A::AtdapL plasmids with the restriction enzymes XbaI and SalI and XbaI and HindIII for the pET30A::CrdapL. Ligate the inserts into the plasmid pBAD33 using T4 DNA ligase to produce the plasmids pBAD33::VsdapL; pBAD33::AtdapL and pBAD33::CrdapL.
		<ol>
			<li>Incubate 50 ng of insert and 20 ng of vector in 1x ligase buffer and 1 unit of T4 DNA ligase overnight at 17 &#176;C. Transform ligation into E. coli Dh5&#945; cells and screen for colonies on LB plates supplemented with 34 &#181;g/ml&#8722;1 kanamycin by incubating 37 &#176;C for 24 hr.20</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cloning of Tyrosine Aminotransferase (tyrB) from A. thaliana for Functional Complementation. 
	NOTE: The complete details regarding the cloning of the cDNA from A. thaliana annotated by the locus tags At5g36160 has been described previously.4 
	<ol>
		<li>Amplify the cDNA by PCR. Use 1 cycle at 94 &#176;C for 2 min, followed by 30 cycles of 94 &#176;C for 15 sec, 60 &#176;C for 30 sec and 72 &#176;C for 2 min. Include 12 pmoles of each primer, 1 mM MgSO4, 0.5 mM each of the four deoxynucleotide triphosphates, and 0.5 ng of template DNA and 1 unit of Pfx DNA polymerase. 
		NOTE: The primers used for the cloning of the At5g36160 are as follows: 
		AttyrB F- 5&#39;-CCCCGAATTCATGGGAGAAAACGGAGCCAAGCGAT-3&#39; 
		AttyrB R- 5&#39;- CCCCGAATTCATGGGAGAAAACGGAGCCAAGCGAT-3&#39;</li>
		<li>Clone the fragment into the plasmid pET30A to produce the recombinant plasmids pET30A::At5g36160 by digesting the PCR fragment with EcoR1 and Sal1; ligate in the fragment into pET30A using T4 DNA ligase as described below.4</li>
		<li>To create the functional complementation plasmid, digest the pET30A::At5g36160 with the restriction enzymes XbaI and HindIII, and ligate the insert into pBAD33 using the same restriction enzyme sites to produce the recombinant plasmid pBAD33::At5g3160 using T4 DNA ligase.
		<ol>
			<li>Incubate 50 ng of insert and 20 ng of vector in 1x ligase buffer and 1 unit of T4 DNA ligase overnight at 17 &#176;C. Transform the ligation into E. coli Dh5&#945; cells and screen for colonies on LB plates supplemented with 34 &#181;g/ml&#8722;1 chloramphenicol by incubating 37 &#176;C for 24 hr.4</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cloning of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-meso-2,6-diaminopimelate Ligase (murE) from V. spinosum for Functional Complementation. 
	NOTE: The cloning of the murE ORF from V. spinosum has been described previously.18
	<ol>
		<li>Amplify the open reading frame by PCR. Use 1 cycle at 94 &#176;C for 2 min, followed by 30 cycles of 94 &#176;C for 15 sec, 60 &#176;C for 30 sec and 72 &#176;C for 2 min. Include 12 pmoles of each primer, 1 mM MgSO4, 0.5 mM each of the four deoxynucleotide triphosphates, 0.5 ng of template DNA and 1 unit of Pfx DNA polymerase. Then clone into the plasmid pET100D to produce the plasmid pET100D::VsmurE. 
		NOTE: The primers used for the cloning of the VsmurE are as follows: 
		VsMurE F- 5&#39;- CACCATGACCATTTTGCGCGATCTTATCGAGGGT -3&#39; 
		VsMurE R- 5&#39;- GTCGACTCACTGACGGTCATCCCTCCTTTGGCGTGC-3&#39;</li>
		<li>To produce the plasmid for functional complementation, digest the pET100D::VsmurE 
		With the restriction enzymes XbaI and Sal1 and ligate the insert into pBAD33 to produce the recombinant plasmid pBAD33::VsmurE using T4 DNA ligase.
		<ol>
			<li>Incubate 50 ng of insert and 20 ng of vector in 1x ligase buffer, along with 1 unit of T4 DNA ligase, overnight at 17 &#176;C. Transform ligation into E. coli Dh5&#945; cells and screen for colonies on LB plates supplemented with 34 &#181;g/ml&#8722;1 chloramphenicol by incubating 37 &#176;C for 24 hr.8</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cloning of relA/spoT (rsh) from Novosphingobium sp for Functional Complementation. 
	NOTE: The cloning of the rsh from Novosphingobium sp. has been described previously.17 
	<ol>
		<li>Amplify the rsh ORF in addition to 599 nucleotides upstream of the initiation start site and 46 nucleotides downstream at the termination site by PCR. Use 1 cycle at 94 &#176;C for 2 min, followed by 30 cycles of 94 &#176;C for 15 sec, 60 &#176;C for 30 sec and 72 &#176;C for 2 min. Include 12 pmoles of each primer, 1 mM MgSO4, 0.5 mM each of the four deoxynucleotide triphosphates, 0.5 ng of template DNA and 1 unit of Taq DNA polymerase. Then clone into the plasmid pCR2.1 to produce pCR2.1::Nsprsh.
		<ol>
			<li>Incubate 1 &#181;l of amplified PCR fragment, 1 &#181;l of pCR2.1 vector, 1 &#181;l of salt solution and 1 &#181;l of water. Incubate at 25 &#176;C for 5 min and 2 &#181;l of ligation into E. coli cells, and screen for colonies on LB plates supplemented with 50 &#181;g/ml&#8722;1 kanamycin by incubating 37 &#176;C for 24 hr. 
			NOTE: The primers used for the cloning of the rsh are as follows: 
			rsh F- 5&#39;- GTTGAAAAACGCCGAATAGC -3&#39; 
			rsh R- 5&#39;- GAGACCTGTGCGTAGGTGGT -3&#39;</li>
			<li>For functional complementation, digest the plasmid pCR2.1::Nsprsh with EcoR1 and ligate the insert into the broad host range pRK290 to produce the recombinant plasmid pRK290::Nsprsh using T4 DNA ligase.
			<ol>
				<li>Incubate 50 ng of insert and 20 ng of vector in 1x ligase buffer and 1 unit of T4 DNA ligase overnight at 17 &#176;C. Transform ligation into E. coli Dh5&#945; cells and screen for colonies on LB plates supplemented with 10 &#181;g/ml&#8722;1 tetracycline by incubating 37 &#176;C for 24 hr.17</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Preparation of Electro-competent Bacterial Cells to Facilitate Transformation
NOTE: The preparation of electro-competent cells is based on the protocol 26 for 1.0 L of culture that can be scaled down to a smaller volume (i.e., 250 ml). Please note that this protocol can be used to make all the strains competent that are described in this manuscript to facilitate FCA.22
<ol>
	<li>Inoculate 50 ml of liquid medium with a single colony into a flask and grow over night, making sure to check the genotype of the mutant before commencing this step (Table 2).</li>
	<li>On day two, inoculate 1.0 L of the appropriate medium (LB for E. coli mutants and PD for Novosphingobium sp) with the 50 ml of the overnight culture, and grow to an OD600 to 0.4-0.6 (log phase) at 30 &#176;C.</li>
	<li>Harvest cells by centrifuging at 5,000 x g for 15 min at 4 &#176;C, decant the supernatant, and resuspend the pellet in 500 ml of sterile ice-cold pure H2O.</li>
	<li>Centrifuge the cells at 5,000 x g for 20 min at 4 &#176;C, decant the supernatant and resuspend the pellet in 250 ml of sterile ice-cold 10% glycerol.</li>
	<li>Centrifuge the cells at 5,000 x g for 20 min at 4 &#176;C, decant the supernatant, and resuspend the pellet in 2.0 ml of 10% glycerol.</li>
	<li>Aliquot the cells by transferring 50 &#181;l into micro-centrifuge tubes and immediately freeze by placing the tubes in a dry-ice-ethanol bath. Store the competent cells at -80 &#176;C for electroporation.</li>
</ol>
3. Electroporation of Bacterial Cells with Complementation Plasmids
<ol>
	<li>For electroporation, add 1.0 &#181;l (10-50 ng) of recombinant plasmid to an aliquot (50 &#181;l) of competent cells and place on ice for 5 min.</li>
	<li>Transfer the mixture via pipetting to an electroporation cuvette and set the electroporation apparatus to the following setting: 25 &#181;F capacitance, 2.5 kV, and 200 ohm resistance and deliver a pulse.</li>
	<li>Add 1.0 ml of recovery media (LB) to the electroporation cuvette and transfer using a pipette to a 15 ml conical tube. Recover with gentle rotation for 60 min in a shaking incubator.</li>
	<li>Plate 100 &#181;l of the culture from the recovery step onto the agar plates to select for transformants by pipetting and spreading using a sterile spreader. 
	NOTE: Make sure to check Table 1 and Table 2 before commencing this step to check the antibiotics and genotypes to make the proper agar plates.</li>
</ol>
4. Transformation of AOH1 to Facilitate Functional Complementation Using L,L-diaminopimelate Aminotransferase (dapL)
<ol>
	<li>For complementation analysis, transform AOH1 with the empty vector (pBAD33), and with the DapL expression vectors in separate transformation events using the electroporation protocol outlined in section 3.0.</li>
	<li>Select transformants by plating on LB agar medium supplemented with 50 &#181;g/ml&#8722;1 DAP and 34 &#181;g/ml&#8722;1 chloramphenicol and 50 &#181;g/ml&#8722;1 kanamycin.</li>
	<li>Test for functional complementation by replica-plating colonies by streaking onto LB medium with 0.2% (w/v) arabinose with and without 50 &#181;g/ml&#8722;1 DAP and 34 &#181;g/ml&#8722;1 kanamycin.</li>
	<li>Incubate plates at 30 &#176;C for 24 hr and observe results. 
	NOTE: The result should show that the mutant is only able to grow on DAP free media only when the dapL gene is expressed in the mutant background when compared to the vector only control.</li>
</ol>
5. Transformation of TKL-11 to Facilitate Functional Complementation Using UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-meso-diaminopimelate Ligase (murE)
<ol>
	<li>Transform the mutant with the empty vector (pBAD33) and with the MurE expressing vector (pBAD33::VsmurE) using the protocol outlined in section 3.0.</li>
	<li>Plate and select transformants on LB agar medium supplemented with 50 &#181;g/ml&#8722;1 thymine and 34 &#181;g/ml&#8722;1 chloramphenicol and incubate the plates at 30 &#176;C for 24 hr.</li>
	<li>Test for complementation by streaking or plating colonies from both the control and the experimental transformations onto two LB medium plus 0.2% (w/v) arabinose and 50 &#181;g/ml&#8722;1 thymine.</li>
	<li>Incubate one plate at 30 &#176;C and the other at 42 &#176;C for 24 hr to visually assess the growth phenotype. 
	NOTE: The results should show that the mutant is able to grow at 42 &#176;C only when the murE gene is expressed in the mutant background compared to the vector only control.</li>
</ol>
6. Transformation of DL39 to Facilitate Functional Complementation Using Tyrosine Aminotransferase (tyrB)
<ol>
	<li>Transform DL39 with either pBAD33 or pBAD33::At5g36160, and select transformants on LB agar plates supplemented with 50 &#181;g/ml&#8722;1 tyrosine, 50 &#181;g/ml&#8722;1 phenylalanine, and 34 &#181;g/ml&#8722;1 chloramphenicol using the protocol outlined in section 3.0.</li>
	<li>Replica-plate colonies on minimal (M9) media with 50 &#181;g/ml&#8722;1 phenylalanine and 50 &#181;g/ml&#8722;1 tyrosine, 50 &#181;g/ml&#8722;1 aspartate, 50 &#181;g/ml&#8722;1 leucine, 50 &#181;g/ml&#8722;1valine, 50 &#181;g/ml&#8722;1 isoleucine, 10 &#181;g/ml&#8722;1 uracil 0.5% (w/v) glycerol, 0.2% (w/v) arabinose. Also replica-plate colonies on plates lacking phenylalanine and tyrosine by streaking the same colony of both plates.</li>
	<li>Incubate plates at 30 &#176;C for 48 hr to observe growth phenotype. 
	NOTE: The result should show that the mutant is only able to grow on phenylalanine and tyrosine free media, only when the tyrB gene is expressed in the mutant background when compared to the vector only control.</li>
</ol>
7. Transformation of Hx699 to Facilitate Functional Complementation of the Hypomucoid Phenotype of Novosphingobium sp. Strain Hx699
<ol>
	<li>Transform wild-type strain Novosphingobium sp. (Rr2-17) and the mutant Hx699 with pRK290 and pRK290::rshNsp in 2 separate transformation events using the transformation protocol outlined in section 3.0.</li>
	<li>Plate the transformations on potato dextrose (PD) agar supplemented with 10 &#181;g/ml&#8722;1 tetracycline, and incubate for at least 24 hr or until transformants appear.</li>
	<li>Streak both transformations in an &#34;X&#34; pattern on fresh PD agar plates and incubate at 30 &#176;C for at least 4 days. Observe the phenotypes of the vector only, and experiment by visually examining the growth phenotype of both plates. 
	NOTE: The result should show that the hypo-mucoid phenotype is complemented when rsh is expressed in the mutant background when compared to the vector only control.</li>
</ol>

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<li>Gan, H. M., Buckley, L., Szegedi, E., Hudson, A. O., Savka, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+an+rsh+Gene+from+a+Novosphingobium+sp.+Necessary+for+Quorum-Sensing+Signal+Accumulation%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">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/pubmed?term=((Genomic+and+biochemical+analysis+of+the+diaminopimelate+and+lysine+biosynthesis+pathway+in+Verrucomicrobium+spinosum%3A+Identification+and+partial+characterization+of+L%2CL-diaminopimelate+aminotransferase+and+UDP-N-acetylmuramoylalanyl-D-glutamyl-2%2C6-meso-diaminopimelate+ligase%5BTitle%5D)+AND+%22Front.+Microbiol%22%5BJournal%5D)">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., Giròn, I., Dobson, R. C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystallization+and+preliminary+X-ray+diffraction+analysis+of+L%2CL-diaminopimelate+aminotransferase+%28DapL%29+from+Chlamydomonas+reinhardtii%5BTitle%5D)+AND+%22Acta+Cryst%22%5BJournal%5D)">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 òn, I., Hudson, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((L%2CL-Diaminopimelate+Aminotransferase+from+Chlamydomonas+reinhardtii%3A+A+Target+for+Algaecide+Development%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Tight+regulation%2C+modulation%2C+and+high-level+expression+by+vectors+containing+the+arabinose+pBAD+promoter%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSambrook%2C+J+%22Molecular+Cloning%3A+A+laboratory+manual%22">Molecular Cloning: A laboratory manual</a>. , 3rd, Cold Spring Harbor Laboratory Press. New York. (2001).</li>
<li>Lugtenberg, E. J., van Schijndel-van Dam, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temperature-sensitive+mutants+of+Escherichia+coli+K-12+with+low+activity+of+the+diaminopimelic+acid+adding+enzyme%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">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/pubmed?term=((Multispecific+aspartate+and+aromatic+amino+acid+aminotransferases+in+Escherichia+coli%5BTitle%5D)+AND+%22J.+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((Escherichia+coli+mutants+deficient+in+the+aspartate+and+aromatic+amino+acid+aminotransferases%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">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/pubmed?term=((A+multispecific+quintet+of+aromatic+aminotransferases+that+overlap+different+biochemical+pathways+in+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22J.+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((Regulation+of+the+stringent+response+in+the+essential+function+if+the+conserved+bacterial+G+protein+CgtA+in+Vibrio+cholera%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci%2C+USA%22%5BJournal%5D)">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/pubmed?term=((Broad+host+range+DNA+cloning+system+for+Gram-negative+bacteria%3A+construction+of+a+gene+bank+of+Rhizobium+meliloti%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci%2C+USA%22%5BJournal%5D)">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/pubmed?term=((Purification+and+enzymatic+characterization+of+a+novel+%CE%B2-1%2C6-glucosidase+from+Aspergillus+oryzae%5BTitle%5D)+AND+%22J+Biosci+Bioeng%22%5BJournal%5D)">Purification and enzymatic characterization of a novel β-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/pubmed?term=((A+key+role+for+ALD1+in+activation+of+local+and+systemic+defenses+in+Arabidopsis%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">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/pubmed?term=((Automatic+prediction+of+protein+function%5BTitle%5D)+AND+%22Celuular+and+Molecular+Life+Sciences%22%5BJournal%5D)">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/pubmed?term=((Complementation+of+the+Arabidopsis+pds1+mutation+with+the+gene+encoding+p-Hyrdoxyphenylpyruvate+dioxygenase%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">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/pubmed?term=((Gene+expression+and+cell+fusion+analyzed+by+lacZ+complementation+in+mammalian+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci%2C+USA%22%5BJournal%5D)">Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells.</a> Proc Natl Acad Sci, USA. 93, 12423-12427 (1996).</li>
</ol>

The authors declare that they have no competing financial interests.

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

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

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 (ΔdapD::Kan2, dapE6) harbors a mutation in the dapE gene and a complete deletion of the dapD gene (<s...

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 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

functional-complementation-analysis-fca-laboratory-exercise-designed

Research

JoVE Journal

Biology

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Bimolecular Fluorescence Complementation

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Measuring Lactase Enzymatic Activity in the Teaching Lab

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

In this protocol, we describe how to make Loewe additivity-based drug interaction measurements for pairwise and three-way drug combinations.

Diagonal Method to Measure Synergy Among Any Number of Drugs

Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Large genetic screens in model organisms have led to the identification of negative genetic interactions. Here, we describe a data integration workflow using data from genetic screens in model organisms to delineate drug combinations targeting synthetic lethal interactions in cancer.

A Data Integration Workflow to Identify Drug Combinations Targeting Synthetic Lethal Interactions

DeepOmicsAE is a workflow centered on the application of a deep learning method (i.e., an autoencoder) to reduce the dimensionality of multi-omics data, providing a foundation for predictive models and signaling modules representing multiple layers of omics data.

DeepOmicsAE: Representing Signaling Modules in Alzheimer's Disease with Deep Learning Analysis of Proteomics, Metabolomics, and Clinical Data

This protocol provides a method for the systematic global optimization of genetically encoded biosensors through automation-assisted genetic library generation and assessment. This is coupled with design-of-experiment methodologies to streamline experimentation and enable the selection of genetic components to tune biosensors to specific design outcomes.

Efficient Sampling of Genetically Encoded Biosensor Design Space Enabled with a Design of Experiments and Automation Workflow

学ぶ機能的相補分析法による生化学的経路の酵素活性評価。変異細胞への遺伝子導入で野生型表現型を回復し、DapL・MurE・RSHなどの代謝酵素のin vivo機能を解明する教育用実験手法です。

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

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