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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

1. Cloning of Diaminopimelate Aminotransferase (dapL) for Functional Complementation.
   1. Amplify the dapL open reading frame (ORF) from the V. spinosum and cDNAs from A. thaliana and C. reinhardtii by ....

Results

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 (

Discussion

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.......

Materials

List of materials used in this article

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