Student project information

For convenience, we have divided the student project information into three parts: Introduction, Questions, and Procedures. Below, you will find the introduction; to view the other sections, please select them from the menu to the left.

Introduction

Conservation Biology spans many aspects of Biology, from Ecology to Genetics.  This is the lab manual for the LABS capstone project in which you will study how edge-effects influence the genetic makeup of sugar maples (Acer saccharum) in an isolated patch of woods.  To do this, you will examine the protein phosphoglucose isomerase (PGI), an enzyme used during the second step of glycolysis.  You will be collecting data from your particular area.  Other individuals and schools will be doing the same for their areas. The LABS program will use your data and that of other participating LABS schools to examine this conservation question on a regional scale.

Note: Materials you will need for each part of this capstone are listed at the beginning of each section. A complete list is in Appendix 1.

The Genetics of Phosphoglucose Isomerase (PGI)

Phosphoglucose isomerase is an enzyme composed of two protein subunits, forming a dimer.   The genetic information of each subunit is contributed by each parent, resulting in a heterozygous or a homozygous genotype, influencing the phenotype (i.e., how each active protein will actually function).  Most protein molecules are slightly negative, making them useful in gel electrophoresis. 

The differing negative charges of different protein molecules will reveal the genotype of the sugar maple samples you collect.  For example, if there are two different forms of a gene (alleles) in your population, there are three possible genotypes (a represents the allele for the faster moving subunit and b represents the allele for the slower moving unit): 

·         Homozygous a (designated as aa), means both parents contributed the a allele at the PGI gene.  These maples only produce the a subunit.

·         Homozygous b (designated as bb), means both parents contributed the b allele at the PGI gene.  These maples only produce the b subunit.

·         Heterozygous (designated as ab) means one parent contributed the a allele and one parent contributed the b allele at the PGI gene.  These maples produce both types of subunits, which combine randomly to produce aa dimers, bb dimers, and ab dimers.   

Your real population of sugar maples may have more than two alleles.
 

Gel Electrophoresis  

In this experiment, you will take samples containing these enzymatic proteins and place them into a polyacrylamide gel, which acts as a matrix through which these proteins travel.  When an electric current is applied to the gels, the proteins will move from the negative end of the gel to the positive end at varying speeds.  The net negative charge of the protein molecules (the charge of the subunits combined) will determine how fast or how slow the protein will move to the positive end of the gel.  Even though the mass and shape of the molecules can influence how the molecules travel through the gel, these aspects can be ignored in this experiment because the net charge is the main determining factor. 

By using gel electrophoresis, you will be able to determine the frequency of the genotypes of the sugar maples, according to which bands appear on the gels after the staining procedure.  The net charge—the charges of both subunits combined—determines how fast the PGI samples will travel through the gel, which determines where the bands on the gel will appear.  Therefore in homozygous samples, the aa dimers should travel closer to the positive end of the gel than the bb dimers. 

However, heterozygous samples will reveal three bands on the gel because all three types of dimers (aa, bb, and ab) are produced.  The band closest to the positive end of the gel contain a group of aa dimers, the band farthest from the edge of the gel contain a group of bb dimers, and the band in the middle are a group of ab dimers, which means its net charge is less negative than the aa dimers but more negative than the bb dimers.  After the electrophoresis run, you will take the results and draw conclusions based on what you studied about edge effects in the workshop lectures.