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1 : overbeek 1.1 <h1>The Initial Attempt to Produce a Metabolic Reconstruction</h1>
2 :    
3 :     A metabolic reconstruction refers to an attempt to infer the metabolic
4 :     machinery of an organism from the sequenced genome and available
5 :     literature. The term was introduced by Evgeni Selkov in his early
6 :     work on the first sequenced genomes. Selkov made available his
7 :     substantial collection of encoded metabolic pathways, and those along with
8 :     existing encodings (most notably the wonderful pathway charts created
9 :     by Gerhard Michal and distributed by Boehringer Mannheim) launched
10 :     numerous efforts to encode the metabolism of sequenced organisms.
11 :     The major effort by <a href=http://www.genome.jp/kegg/>KEGG</a> has
12 :     become, perhaps, the most well known, and is what the SEED effort has
13 :     tended to utilize.
14 :    
15 :     <p>
16 :     Different groups have created slightly differing notions of what is
17 :     meant by <i>metabolic reconstruction</i>. Within the context of this
18 : overbeek 1.2 course, we draw the following distinctions:
19 : overbeek 1.1 <ol>
20 :     <li>By an <b>informal metabolic reconstruction</b> we refer to
21 :     <ul>
22 :     <li>
23 :     taking the genes of an organism and dividing them into small groups
24 :     that each perform some well-defined cellular function,
25 :     <li>
26 :     identifying the overall function of each of these groups, and
27 :     <li>
28 :     attaching to each gene a list of the abstract functions implemented by
29 :     each gene.
30 :     </ul>
31 :     With informal metabolic reconstructions, it is common to include not
32 :     only metabolic subsystems (i.e., pathways), but nonmetabolic
33 :     subsystems, as well.
34 :     <li>
35 : overbeek 1.2 By a <b>formal metabolic reconstruction</b> we refer to a detailed
36 : overbeek 1.1 encoding of the metabolic reaction network of the organism.
37 :     </ol>
38 :     That is, the informal reconstruction attempts to represent as much of
39 : overbeek 1.2 the cellular machinery as possible. It provides a solid foundation
40 :     from which the formal metabolic reconstruction can be based. However,
41 :     the informal metabolic reaction has substantial by itself. There are
42 :     many aspects of the phentotype that can be inferred by just
43 :     qualitative reasoning based upon the presence or absence of specific
44 :     subsystems or functional roles. Further, many aspects of the
45 :     biochemistry (e.g., "missing genes") can be analyzed from just the
46 :     perspective of the informal metabolic reconstruction.
47 :     <p>
48 :     The formal is usually
49 :     limited to just metabolic reactions (and those reactions involving
50 : overbeek 1.1 generation or degradation of polymers are normally left out). The
51 :     output of a formal metabolic reconstruction will include detailed
52 :     encodings of both the reactions and the compounds that appear in the
53 : overbeek 1.2 metabolic network.
54 :     >p>
55 : overbeek 1.1 These distinctions are ours, and are not commonly used.
56 :     We consider them unimportant, but useful.
57 :    
58 :     <p>
59 :     In this section of the course, we are asking the student to build both
60 :     an informal and a formal metabolic reconstruction for some sequenced
61 :     organism. Clearly this is an ambitious task. It would have been
62 :     largely impossible to do anything significant 10 years ago, but with
63 :     the new tools we believe that this effort can be quite productive as
64 :     an amazing crash course in biochemistry and microbial physiology.
65 :    
66 :     <p>
67 :     Rather than break this part of the course up into weekly assignments
68 :     (at least for now), we list the detailed steps we would like the
69 :     student to work through.
70 :    
71 :     <p>
72 :     We are going to suggest that each student be assigned a distinct
73 :     organism (alternatively, groups of students can work jointly on a
74 :     single organism). We sugesst choosing an organism that fulfills the
75 :     following criteria:
76 :    
77 :     <ul>
78 :     <li>It should be a small to moderately large sequenced, prokaryotic genome
79 :     (450-2500 genes).
80 :    
81 :     <li>It should be a genome for which metabolic reconstructions have not
82 :     already been done or are known to be in progress.
83 :    
84 :     <li>It should be in the public domain,
85 :    
86 :     <li>The genome should be included in both the KEGG collection and in
87 :     the SEED collection.
88 :     </ul>
89 :    
90 :     <h2>Steps in the Process of Developing an Informal Metabolic Reconstruction</h2>
91 :    
92 :     <h3>Getting summaries of what is in the genome</h3>
93 :    
94 :     First, you should get two estimates of what cellular machinery is
95 :     present in the organism:
96 :     <ol>
97 :     <li>You should get a list of the subsystems with operational variants
98 : overbeek 1.2 from a SEED installation.
99 :     The easiest way to do this involves starting from the first page
100 :     of the SEED, asking for <b>Statistics</b> for the genome you are
101 :     working on, and then (near the bottom of the page) clicking on <b>Show
102 :     subsystems</b>.
103 :    
104 :     Note that the subsystems and genes that you
105 : overbeek 1.1 get back may include both well-curated subsystems and
106 :     poorly-constructed subsystems.
107 :     <li>You should get colored versions of the KEGG maps (showing which
108 :     functions are believed to be present in the genome).
109 :     </ol>
110 :    
111 :     <h3>Begin with the Common Machinery</h3>
112 :    
113 :     There is a subset of the cellular machinery that will be present in
114 :     some form in whichever genome you picked. The ribosomal RNA,
115 :     ribosomal proteins, tRNAs, tRNA synthetases, and so forth must all be
116 :     there. Look through the set of subsystems that are present, decide
117 :     what aspects appear to be essential machinery relating to
118 :     transcription and translation, and begin with that. Create a detailed
119 :     summary of which topics you have selected, which variants exist, and
120 :     which genes implement those variants. Which rRNAs and tRNAs exist?
121 :     How many copies of the rRNA cluster exist?
122 :    
123 :     <h3>Studying Amino Acid Synthesis</h3>
124 :    
125 :     Next, we suggest <i>amino acid metabolism</i>, or even more restricted
126 :     <i>the synthesis of amino acids</i>. Identify which of the KEGG maps
127 :     address this section of metabolism, and then which subsystems from the
128 :     SEED are relevant. Now prepare a list of the amino acids that can be
129 :     synthesized, along with the starting point in each case. Make sure
130 :     that you compose a detailed list of outstanding questions.
131 :    
132 : overbeek 1.2 <h3>Synthesis of Nucleotides</h3>
133 : overbeek 1.1
134 : overbeek 1.2 We suggest that you next turn your attention to synthesis of nucleotides.
135 :     Locate the appropriate KEGG charts and the relevant
136 : overbeek 1.1 subsystems. Again, summarize the situation, along with outstanding
137 :     questions.
138 :    
139 :     <h3>Systematically Work Through the Central Cellular Machinery</h3>
140 :    
141 :     Between the SEED hierarchy, the KEGG maps, and the numerous examples
142 :     of metabolic reconstructions published in genome papers, you have
143 :     numerous examples of the basic components of a functional hierarchy.
144 :     You should choose a reasonable organizational style and produce
145 :     an HTML document comprising your best effort at an informal metabolic
146 :     reconstruction.
147 :    
148 :    
149 :     <h2>The Basic Steps in Building a Formal Metabolic Reconstruction</h2>
150 :    
151 :     You should begin by studying exactly how Bernhard Palsson and his team
152 :     have built formal metabolic reconstructions:
153 :     <ul>
154 :     <li><a
155 : overbeek 1.2 href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12952533&query_hl=1">Escherichia coli</a>,
156 : overbeek 1.1 <li><a
157 :     href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15752426&query_hl=2"><i>Staphylococcus
158 :     aureus</i></a> and
159 :     <li><a
160 :     href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12142428&query_hl=5"><i>Helicobacter
161 :     pylori</i></a>
162 :     </ul>
163 :    
164 :     You are being asked to construct a list of several hundred reactions,
165 :     where each reaction includes precise substrates, products and
166 :     (possibly) a required enzyme.
167 :    
168 : overbeek 1.2 <h3>Begin from the Informal Metabolic Reconstruction</h2>
169 : overbeek 1.1
170 :     You should begin from the informal metabolic reconstruction and
171 :     accumulate the reactions and compounds implied by the operational
172 : overbeek 1.2 variants of the subsystems.This can be done by starting from the first page
173 :     of the SEED, asking for <b>Statistics</b> for the genome you are
174 :     working on, and then (near the bottom of the page) clicking on <b>Show
175 :     reactions</b>.
176 :     This tool produces an initial estimate of the reaction set.
177 :     <p>
178 :     This initial set is far from complete and some of the reactions
179 :     presented will be encoded improperly. Before continuing let us just
180 :     ponder what a "complete and accurated formal metabolic reconstruction"
181 :     would contain:
182 :     <ul>
183 :     <li>It would contain all of the metabolic reactions. For many
184 :     purposes it might be useful to exclude synthesis and degradation of
185 :     polymers. On the other hand, for whole organism modeling, it becomes
186 :     necessary to estimate the compounds that can be transported into and
187 :     out of the cell. This is quite difficult. For the purposes of this
188 :     class you should ignore the reactions relating to polymers, and you
189 :     should ignore the issue of exactly what can be transported.
190 :    
191 :     <li>The reaction set should not contain <i>class reactions</i> (those
192 :     in which the substrates and products are not specific).
193 :    
194 :     <li>You can construct a list of compounds that must be
195 :     present based on this incomplete reaction set. To get the initial
196 :     approximation of this set, ...
197 :    
198 :     <li>From the list of reactions and compounds, you should be able to
199 :     produce a set of compounds that exist as substrates for one or more
200 :     reactions, but not as products. This computation cannot be done
201 :     accurately without knowing which reactions are reversible. This can
202 :     be done using ...
203 :    
204 :     <li>Finally, you should produce a list of compounds that exist as
205 :     products, but not as substrates. This can be done using ...
206 :     </ul>
207 :    
208 :     <p>
209 :     For the purposes of this class, construction of this initial, crude
210 :     formal metabolic reconstruction is both the best you can do and a
211 :     major achievement. To refine it into a useful and accurate summary of
212 :     the metabolism of the cell is something that a person might work a
213 :     lifetime on.
214 :    
215 :     <h2>Summary</h2>
216 :    
217 :     The object of this portion of the class will be to development both an
218 :     initial <b>informal metabolic reconstruction</b> and a <b>formal
219 :     metabolic reconstruction</b> for some specific organism. If you can
220 :     successfully achieve this, you will have done something that was
221 :     almost impossible even a few years ago. If you study and reflect on
222 :     what you accomplish, it will form a starting point for deepening your
223 :     understanding of microbial physiology and biochemistry.

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