5. BioBRICKS

The goal of our project was to investigate and demonstrate the feasibility of polypeptide assembly based on modular nanoBricks. Potentials of this approach are vast (see Discussion and Vision) and for the development of applications it is essential to have available a large collection of “nuts and bolts” to assemble polypeptide nanostructures. We produced alltogether more than 100 BioBricks, which comprise a significant number of different natural as well as designed coiled-coil forming segments as well as different polypeptide oligomerization domains. In addition we prepared several “functional polypeptides”, which provide additional useful features to the material, such as different biological activities (antimicrobial peptide, growth factors, cell attachment motifs…), optical properties, enzymatic activity...

On the other hand we extended the BioBrick standard by introducing sites that allow extension of peptide linker sequences. Length of the linkers between polypeptide domains is crucial to determine the accessible geometry of the assembly, and our extended standard provides a tool to extend the length of a linker by any required length in increments of two residues. This task, particularly concerning small extensions would otherwise require the preparation of a new domain construct.

Development of BioBricks

Linker-extension standard

A new developed linker-extension standard is also described in detail under BBF RFC37(link to http://dspace.mit.edu/bitstream/handle/1721.1/46705/BBFRFC37.pdf?sequence=1) To improve the efficacy of cloning, we designed a NEW BioBrick standard that enables simplified and efficient linker extension between protein domains (link to Figure 0) and at the same time preserve the characteristics of the most extensively used BioBrick standards. Two variations of linker-extension standard were designed. Both variations contain 5' and 3' cloning restriction sites EcoRI, PstI, NotI, XbaI and SpeI characteristic for BBa standard (Figure 1 - link). Additionally, core restriction sites NgoMIV, AgeI, XmaI, BspEI are added. These restriction sites are used for linker extension and their positions differ among two variations of linker-extension standard. The position and the usage of these core restriction sites determine amino acid residues incorporated in the linker between protein domains (Figure 2 - link).

Figure 1 Figure 1: Schematic presentation of basic elements of two linker-extension standards also named BB-NIC-II and BB-NIC-III vectors.

Figure 2 Figure 2: Schematic presentation of linker extension using both variations of linker-extension standards. A basic BRICK is re-cloned into the linker-extension standard using suitable restriction sites to obtain either Thr-Gly, Ser-Gly or Pro-Gly extensions.

Linker extension is not limited to the addition of only two amino acid residues between protein domains (Figure 3 – link). Each round of cloning into the linker-extension standard incorporates two additional amino acid residues. The step of re-cloning could be repeated indefinitely.

Figure 3 Figure 3: A schematic presentation of repetitive linker extension. Similar strategy could be used to incorporate other linker amino acids (link). With each round of cloning the insert gains two additional amino acids. Type of linker amino acids is defined with way of cloning (restrictions of vectors).

Detailed Cloning Instruction using the linker-extension standard A or the linker-extension standard B, BioBrick-NIC-III, are described in Figure 4 (link) and Figure 5 (link).

Figure 4 Figure 4: Schematic presentation of three different cloning strategies in multiple-cloning site of BB-NIC-II vector. Note: Each type of cloning leaves different extension of amino acids that could be used to extend scar/linker between parts after in-frame parts assembly.

Figure 5 Figure 5: Schematic presentation of four different cloning strategies in multiple-cloning site of BB-NIC-III vector. Note: Each type of cloning leaves different extension of amino acids which could be used to extend scar/linker between parts after in-frame parts assembly.

Assembly strategy joining two parts/bricks is depicted in Figure 6 (link). A detailed description is described also under BBF RFC37 (link).

Figure 6 Figure 6: A schematic presentation of parts assembly. Each of two BRICKs is cut with suitable restriction enzymes and three point ligation into vector (with ccdB domain) cut with EcoRI and PstI is performed. Note: Also the standard BBa assembly strategy could be used.

Compatibility with other BBa standards The new linker-extension standard is fully compatible with BBa and other BBa compatible standards. Each part suited for cloning into standard BioBrick vector could be cloned also into BB-NIC vectors. The part suitable for standard Biobrick vector could be cloned into BB-NIC vectors into XbaI, SpeI restriction sites with some limitations. No linker extension strategy is applicable for those parts. Transfer of parts from linker-extension standard to BBa vector could be achieved in two steps (Figure 7-link).

Figure 7 Figure 7: A schematic presentation of part transfer from BB-NIC into BBa and alike vectors. Two steps of cloning are required for directional cloning. Step 1: Ligation of part from BB-NIC into BBa both cut with XbaI/PstI. Step 2: Ligation of part from intermediate into BBa both cut with EcoRI/SpeI.

nanoBRICKs[PRO] vectors

The main goal of iGEM project was to create versatile two or three dimensional protein structures. Shapes of these structures depend not only on protein domains characteristics but also on the length of the linker connecting protein domains as schematically presented in Figure (link).

Figure 1

First challenge of our iGEM team was a development of a simple and efficient cloning system for extending a linker between interconnected protein domains. The need for that is the fact that almost all parts were ordered by gene synthesis adapted for cloning into BioBrick standards. To avoid ordering same protein domain with different linker extensions, the NEW BioBrick standard, named the linker-extension standard was designed. As basics, the BBF RFC 25 (fusion protein (Freiburg) BioBrick assembly) standard was used. The modifications in multiple-cloning site enable (i) in-frame fusion, (ii) friendly scar and (iii) simple extension of linker between parts. A comprehensive description of the linker-extension standard (link to 5.1.1 Linker-extension standard) is under BBF RFC37 (link to http://dspace.mit.edu/bitstream/handle/1721.1/46705/BBFRFC37.pdf?sequence=1).

Functionalised linker-extension standard, nanoBRICKs[PRO] Although most promoters, ribosomal binding sites, tags and terminators are currently specified as separate parts in the Registry, we are now moving to a new design in which these elements are included within the vector. Our working hypothesis is that the new design will reduce the likelihood of unexpected functional composition problems between promoter / terminator and coding sequence.

Several types of nanoBRICKs[PRO] vectors were constructed (Figure 2 link) with different 5' and 3' sequences coding specific promoters, terminators and coding sequences: KSI domain, his-tag, etc.

Figure 2

Table of functionalised nanoBRICKs[PRO] vectors