HEARTBEAT Features

Selecting initial parameters

In the first page of our GUI the user is requested to enter basic parameters determining the primary assembly of the promoter. The inexperienced user has to provide only the final length of the construct assuming that the user will take the CMV core promoter from Part:BBa K203113 to complete his/her construct. In this case the length of the core promoter has a length of 80 bases and the transcription start site is -20 bases upstream regarding the end of the CMV promoter. Users who already possess a prefered promoter have the opportunity to specify its length and the location of the TSS in the advanced mode. The user is not able to enter values over 1000 base, since the frequency distributions for each transcription factor binding site (TFBS) stored in the HEARTBEAT-database (DB) scope the distance from 0 to 1000 bases upstream of the TSS.

Selecting main and auxiliary transcription factors

As soon as the user submits his/her selected parameters, he/she is forwarded to the selection of the main transcription factor (TF), whose binding site is supposed to be integrated into the construct. The user can choose between 144 different binding sites. He/she should be aware beforehand of a mechanism to exclusively induce the pathway where the TF is involved in. For further recommendations and further information to build up an experiment fulfilling this requirement please read links. Once a primary TF is chosen two plots become visible. The first one shows the frequency histogram deduced from the HEARTBEAT-DB. The solid red line depicts the probability density function smoothing the TFBS distribution. The position of the vertical red line is stored in the background and used later in the final promoter assembly. After the sequence is automatically assembled the user will have the opportunity to modify the sequence by his own account. In case of multiple maxima the user can manually introduce further binding sites influenced by the pdf. In the second plot the frequencies of all co-occurring TFBS can be seen. The five most frequent occurring TFBS can be chosen in within the next frames. Again the maxima of the respective pdf are stored by the program.

Selecting or entering a consensus motive

When all favoured TFBS are chosen, the user is asked to either enter a known binding motive or select one out of the calculated possibilities. The selection comprises for each consensus matrix found in the Transfac database for the chosen TFBS one possible binding motive. Therefore all half defined letters from the “Ambiguity Code” (e.g. M, R, W etc.) are replaced by either A, C, G or T in random process. This might lead to different selection each time this function is used. To determine the quality of a particular TF binding motive, we recommend to test the sequence with the open-source program TRAP and hence to use only binding motives with a high binding affinity. The selected or entered motive is finally transmitted with all other earlier defined parameters to the crucial sequence assembly algorithm.

The assembly algorithm

The assembly algorithm represents the central program in the HEARTBEAT-GUI. As input parameters the algorithm needs the absolute promoter length, the core-promoter length, the TSS position relative to the end of the core-promoter, the chosen binding motives and their distance to the TSS. In an iterative process the algorithm attempts to locate first the main TFBS and then the auxiliary TFBSs at the position of their pdf-maxima. If the consecutive TFBS overlaps with the preceding one, the consecutive TFBS is randomly moved 1 base either to the left or to the rightas long as a valid position is found. Every repositioning done by the algorithm can be read in a warning message in the output window. The final sequence is transferred in the end into a TyneMCE open source web-editor. Within this editor, the user can manually modify its sequence. If the user wants to introduce new binding sites, he/she has to highlight the sight in a different colour in order to enable the program to recognize the site.

Adding spacer sequence to the construct

By pushing “add spacer sequence” a small perl program introduces every chosen TFBS into a deposited random sequence at the pre-defined positions. From this sequence every restriction site used in any BioBrick standard has been removed. Furthermore we also eliminated also every TFBS detected by the Transfac Match tool assuming the sequence to be TFBS free.

Test for restriction sites

With this feature the user is enabled to test his/her final sequence for every restriction site used in BioBrick formats. This program addresses the problem of newly evolving restriction sites at the cutting sites between random sequence and biding motive. The restriction site is manipulated systematically by altering single bases without touching the original binding motive.