"
The registry of standard biological parts is rapidly expanding. Since 2003 more than 15000 BioBrick parts were added by iGEM teams and labs from all over the world, making the life of a synthetic biologist easier and easier with each passing year.
The increase of BioBrick parts – especially of the composite parts – could facilitate fast cloning of the construct of interest. Unfortunately, optimizing your cloning strategy by searching the registry for useful composite parts is laborious and time consuming. Even though the search is time consuming it could be worth doing, since reducing the amount of cloning steps will significantly decrease the amount of time needed for the actual cloning thereby giving you more time for whatever else needs to be done.
That’s why this year’s Wageningen UR team set out to make a program, which automatically creates the most efficient cloning strategy for your construct of interest. The program developed is called “The Constructor” of which the general user interface is seen below.
Mode of action:
The constructor works as follows: for example, you want to create the following hypothetical construct of 1-2-3-4-5-6. A number in this construct represents what we call a functional genetic module (FGM). We classify a FGM as followed: a FGM starts with a promotor and ends with a terminator, the parts in between can vary. For example, the following subparts (R0062-B0034-E1010-B0010-B0012) combined will form a functional genetic module, because it starts with R0063 - which is the lux pL promoter and it ends with B0010-B0012- which is the rrnBT1-T7TE terminator. However, R0062-B0034-C0062-B0034-C0061-B0010-B0012 would also classify as a FGM, because it still starts with a promoter and ends with a terminator.
The hypothetical construct consists of 6 of these FGM’s. Because each FGM can function on its own due to the promotor and terminator, it doesn’t matter where it is in your construct. So you can just scramble the sequence of the FGM’s of the hypothetical construct, to for example 135264 or 125643. This won’t have any adverse effects on the molecular workings, due to the promotors and terminators.
For this hypothetical construct there are 720 (6 faculty) different combinations of arranging the functional modules. The rearranging of the sequence of the functional modules is critical for optimizing the cloning strategy due to the following: lets say we use the previous mentioned functional modules R0062-B0034-E1010-B0010-B0012 (1) and R0062- B0034-C0062-B0034-C0061-B0010-B0012 (2). With these two modules two combination can be made namely 1-2 and 2-1, as seen below.
(1-2) R0062-B0034-E1010-B0010-B0012-R0062-B0034-C0062-B0034-C0061-B0010-B0012
(2-1) R0062-B0034-C0062-B0034-C0061-B0010-B0012- R0062-B0034-E1010-B0010-B0012
With these two combination we search the registry for composite parts which could be used to construct the constructs with. Let’s say you will get the following composite parts for the two constructs:
(1-2)
R0062-B0034-E1010
B0010-B0012
R0062- B0034-C0062
B0034-C0061-B0010-B0012
(2-1)
R0062-B0034-E1010
B0010-B0012
R0062- B0034-C0062
B0034-C0061-B0010-B0012
R0062-B0034-C0062-B0034-C0061-B0010-B0012- R0062-B0034-E1010
As you can see the majority of composite parts found could be used for both construct, however for the construct 2-1 you find an additional composite part namely.
R0062-B0034-C0062-B0034-C0061-B0010-B0012-R0062-B0034-E1010
This composite part combined with the B0010-B0012 part will yield the desired construct. Thus by rearranging the sequence of these functional modules 1 and 2 you effectively reduced the amount of cloning as seen at 1-2 arrangement from 3 cloning steps to 1 cloning step in the 2-1 arrangement.
Furthermore, the program implements a few filters for part quality, part availability and part experience. With these filters only parts containing the criteria which you selected are taken from the database and subsequently used in optimizing your cloning strategy.
The zip file containing the program, the manual and the source code can be downloaded [http://sourceforge.net/p/theconstructor here]. After unzipping the zipfile read the manual for more information about using The Constructor.
Validation of “The Constructor”.
For the validation of “The Constructor” we contacted the following iGEM teams, with the request if we could test their cloning strategy. The results are depicted in the table below.
