= Practical 2 =
== Building a model from MetaCyc ==
Our aim is to construct a model of the Entner-Doudoroff pathway of glycolysis in the natural ethanol-fermenting organism ''Zymomonas mobilis''.   If time permits, we will later use this as a basis to explore whether the substrate  range of this organism could be expanded by metabolic engineering. For  some background to this, you can find two articles [[http://dx.doi.org/10.1016/j.jbiotec.2013.02.014|here]] and [[http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00042/full|here]].

For this, we will use the !ScrumPy module '''!PyoCyc'''  to extract the required reaction equations from a local copy of the  [[http://metacyc.org/|MetaCyc]] database, starting from a list of the enzyme EC numbers.  We  will write the reactions into a !ScrumPy file, which can then be loaded  into !ScrumPy and investigated.  Of course, this automatically-generated  model will not immediately work, for reasons such as different  identifiers being used for the same metabolite in different reactions.   In addition, the reaction list will contain reactions that will not take  place in connection with the Entner-Doudoroff pathway because their  substrates are not available in the cell.

Your  task is to get the model to work so there is a viable route from  glucose to ethanol, and to condense it so that only relevant reactions  are present.

=== Step 1 ===
 1.  Download, by right-clicking on its link, the !ScrumPy file [[http://mudsharkstatic.brookes.ac.uk/Nepal2016/P2/Zymomonas.spy|Zymomonas.spy]] and save it in the directory you are going to use for this exercise.


 1. Open  the model file with !ScrumPy.  Note that there are some generic  components of the model here that we are giving you as a starting point:

  1. An ''Include'' directive that is currently commented out but which will be needed to incorporate the reactions you are going to find.

  1. A  statement that WATER and PROTONS are regarded as external,  freely-available species.  Balancing every last PROTON is hard, so we're  not going to struggle with that yet.

  1. A  set of transporter definitions that state that glucose (GLC), ethanol  (ETOH) and carbon dioxide can exchange between the metabolism and the  environment.

  1. Two  reaction definitions that cannot be retrieved from their EC numbers in  the !MetaCyc data files, even though they are present.



=== Step 2 ===
 1. Open !PyoCyc and load the !MetaCyc database by typing the following in your !ScrumPy window:

 {{{
from Bioinf import PyoCyc
db = PyoCyc.Organism()
}}}

 Note that though you've loaded the generic !MetaCyc, you could in principal load any [[http://biocyc.org/|BioCyc]] format organism database.


 1. Copy this list of EC numbers for the Entner-Doudoroff pathway:
 {{{
ecs=['EC-2.7.1.2', 'EC-3.1.1.31', 'EC-4.2.1.12', 'EC-4.1.2.14','EC-1.1.1.44',  'EC-1.2.1.12', 'EC-2.7.2.3', 'EC-5.4.2.11', 'EC-4.2.1.11', 'EC-2.7.1.40', 'EC-4.1.1.1', 'EC-1.1.1.1']
}}}


 1. You can see the set of reactions associated with each EC number with the following code:
 {{{
for e in ecs:
        for r in db[e]:
                print e, "\n", r.AsScrumPy()
}}}

 Here, each EC number in turn is used as a key, ''e''  into the database, which returns a set of reaction records.  The method  !AsScrumPy formats the record as required for the input file.


 1. You need to generate an input file though, so enter the following:
 {{{
spy=open("ZmED.spy","w")
spy.write('# Entner Duodoroff pathway\n')
for e in ecs:
    str = "# " + e +"\n"
    for r in db[e]:
            spy.write(str)
            spy.write(r.AsScrumPy())

spy.close()
}}}

  . After opening  the file, ZmED.spy, the first write statement puts an identifying  comment in.  Then for each EC number, a comment is inserted giving the  EC number, since this often doesn't appear in the reaction name.


== Step 3 ==
 1.  Un-comment the ''Include''  directive in the Zymomonas.spy file to load your ZmED.spy and recompile  the model using the drop-down ScrumPy menu option. Find out if there is  a potential pathway through it by calculating the null space of the  stoichiometry matrix:

 {{{
ns = m.sm.NullSpace()
ns
}}}

  . Two windows should  now be open, one for Zymomonas.spy and one for ZmED.spy.  However, the  null space is empty, in other words the system defined by this model  cannot support a stead-state flux in any of its reactions.


 1. The common possibilities for the lack of a viable route where there  should be one are either missing reactions, or, when all the reactions  are present,  inconsistencies in metabolite definitions break the  connectivity.  Looking for dangling ('''orphan''') metabolites can point to both of these issues:

 {{{
m.sm.OrphanMets()
}}}


 1. If  the network connectivity is being broken by inconsistent metabolite  naming, edit ZmED.spy, save it, and recompile the model.  Then check the  null space and orphan metabolites again.

 1. Once you get one or more null space vectors, you can compute the  elementary modes of the model and the stoichiometry of their overall  reaction to check whether you have the exected conversion of glucose to  ethanol.

 1. The model can now be reduced by removing reactions that will always be  dead.  Calculate the enzyme subsets of the model; all the dead reactions  will be in the "dead" subset.  Rather than deleting the reactions  immediately, remove them from the model by placing a comment marker,  "#", at the start of each line belonging to an unwanted reaction.   Recompile the model and check that you have not broken the network.

 1. How many reactions are left in ZmED.spy when you have inactivated all the dead ones?

== Step 4 ==
Time permitting, go to [[http://mudshark.brookes.ac.uk/Meetings/Nepal2016/Prac2supp|the next part]].