Practical 4

Analysing a genome scale metabolic model of A. thaliana

In this practical we (you !) will be replicating some the analysis that was discussed in the previous lecture. In order to do this you will need to download the files associated with the model:

1. Download the file AraGSM.tgz into the area in which you have been using for your other practicals.
2. This is a compressed archive file and you will need to extract the files before they can be used:

   •  $ tar -zxf AraGSM.tgz 

3. This will generate a directory, A.thaliana, containing two sub-directories: Model and Analysis. Model contains the model definition files and an additional python module (in Model/Tools). Analysis contains the python modules you will need for this practical.
4. For the sake of the practical we have made a few simplifications and the model and results will not be identical to those in the lecture. the aim of the practical is to illustrate the techniques used.

Part A:

Investigating the effect of knocking out the Calvin cycle enzymes from GSM of A. thaliana

1. cd into Analysis/Knockouts

2. Start ScrumPy and the load the model as before.

3. Import the KnockOut module.

4. This defines a single function also called KnockOutEffects that returns a dictionary recording the impact of removing each reaction from the model (relative change in objective value)

   1    >>> import BuildLP
   2    >>> lp = BuildLP.BiomassLP(m)
   3    >>> lp.Solve()
   4    >>> wild_type_solution = lp.GetPrimSol()

Observe the fluxes of the Calvin Cycle enzymes

1. Provide the model, reaction to be knocked out and lp}} as argument to the  function {{{KnockOutEffects

2. Store the solution as mutant_solution

   1        >>> mutant_solution  = KnockOut.KnockOutEffects(m, 'SEDOHEPTULOSE-BISPHOSPHATASE-RXN_Plas', lp)

1. Investigate the differences in wild_type_solution and mutant_solution and interpret the results.
2. Similarly , find the reactions that were inactive as a effect of knock out
3. Identify the reactions that have different flux values as compared to wild type and interpret the results.

   1       >>> from Util import Set
   2       >>> new_active_reactions = Set.Complement(mutant_solution, wild_type_solution)
   3       >>> inactive_reactions = Set.Complement(wild_type_solution, mutant_solution)
   4       >>> for rxn in Set.Intersect(mutant_solution, wild_type_solution):
   5       >>>      if abs(mutant_solution[rxn] - wild_type_solution[rxn]) > 1e-5:
   6       >>>            print rxn, mutant_solution[rxn], wild_type_solution[rxn]

1. Repeat Step 5-8 for the FBPase, PRK, G3Pdh and SBPas,  FBPase dual knock out mutants.

Part B : Analysing the response of GSM of A. thaliana to varying input of photon flux

1. Change directory to the relevant area:

   • $ cd A.thaliana/Analysis/LightScan

2. Start ScrumPy and load the model:

   1  >>> m = ScrumPy.Model("../../Model/AraTopLevel.spy")

(If you wish to avoid a bit of typing, leave the model name blank and use the file selector to find the model file instead.)

1. Examine the files that are now presented - how much can you recognise from previous work in this course.
2. Create an LP object with the GSM of A.thaliana as argument (see previous LP exercise for details on how to do this).

3. Set the objective to minimisation of all fluxes. Since minimisation is the default direction, all you need to do is to use the lp.SetObjective() method with all reactions in the LP object as argument, which can be obtained as lp.cnames.values()

4. Use the dictionary of biomass fluxes as fixed constraints.

   1       >>> import BuildLP
   2       >>> fd = BuildLP.biomass_dictionary
   3       >>> lp.SetFixedFlux(fd)

1. Try to solve the LP and make sure the number of reactions in the solution is non-zero

2. You will scan the photon uptake reaction by fixing its flux to a set of value in a linear range, specifically 50 points evenly distributed from 0 to 1. To do this, first generate a list of these values (Use the knowledge from exercise of the python practical).

   1.Define a dictionary (this will be referred to as sol henceforth) where you will collect the LP solutions.

   1.Write a for loop over the list of values generated above. Inside the loop, set the constrain on the photon uptake reaction (Photon_tx) equal to the loop variable, solve the LP, and collect the solution in sol created above (you can use the loop variable as key)

3. You will now analyse the LP data. In order to collect the names of all reactions that appear in any of the solutions, create an empty dictionary and run a for loop to update it with the reaction names from the solutions generated previously, e.g.

   1     >>> reacs = {}
   2     >>> for sol in collection:
   3            reacs.update(sol_collection[sol])

1. Import the module DataSets from Data. Create an instance of the DataSet class, give the keys of the reaction name dictionary just created to the argument with keyword ItemNames, as such:

   1       >>> ds = DataSets.DataSet(ItemNames=reacs.keys())

1. The names provided will be column names in data set created. The DataSet class is a subclass of the ScrumPy matrix class, as are the stoichiometry matrices that you have looked at before, so much of the structure and properties of data sets will be similar.

1. Update the data set with the LP solutions so that each row corresponds to a fixed photon flux. Conveniently, there is a DataSet method, called UpdateFromDic(...), that updates the data set with a dictionary if the keys can be found in the column names of the data set and the values are numbers. Write a for loop over the collection dictionary and update the data set with each solution.

1. Since we are interested in reactions that change flux as a response to changes in photon flux, we need to identify these reaction in the data set. We will do this by looking at the aboslute difference between the smallest and largest flux through each reaction in the set. Use a for loop over the column names of the data set (if the data set is named as ds this is ds.cnames). For each column name get the associated list of values (use the method ds.GetCol(c) with c being the name of column, i.e. if you are in a loop, the loop variable); calculate absolute of the difference between the maximum and minimum flux value in the list (we can use the built-in functions abs(), max(), and min() for this), if this difference is above a fixed threshold the reaction (i.e. the loop variable) will be stored. Here is the code:

   1          >>> changing = []            # list to store reactions with changing flux
   2          >>> lim = 1e-7               # flux threshold
   3          >>> for c in ds.cnames:
   4                 col = ds.GetCol(c)
   5                 if abs(max(col) - min(col)) > lim:
   6                     changing.append(c)

1. Use the plotting methods of DataSet to look at the flux responses of the changing reactions. Set the x-axis as the photon flux with the method ds.SetPlotX(...), where the argument is a column name in the data set ds, here the photon transporter (Photon_tx) (note that ds.SetPlotX(...) only initialises the plot internally, you will not see the plot until data is added). Add columns to the plot using the method ds.AddToPlot(...), where the argument is either a string (name of a column), or a list (of column names). Similarly, you can remove column from the plot using the methods ds.RemoveFromPlot(...) and ds.RemoveAllFromPlot(). You can check all the functions of the dataset dir(ds).