PARTS OVERVIEW

In order to accomplish the functions that the Beetector system requires, Team UAlberta had to design new BioBricks, based on previous publications, that access and expand the functionality of M13 bacteriophage particles and chromoproteins. Our collection (see Parts Table) provides the tools for the post-translational conjugation of two peptides using the SortaseA enzyme from Staphylococcus aureus. Our collection includes the tags recognized by SortaseA as basic parts, and using these we have designed composite Biobricks that can be used to produce tagged reporter proteins and a tagged M13 bacteriophage coat protein. These composite parts will allow the conjugation of the M13 bacteriophage coat protein to the visual reporters described, allowing for the modification and engineering of M13 phage particles post-translation. This strategy of M13 decoration has been successfully used before and our collection aims to bring this toolset to the Registry. These parts can be used beyond M13 modification, as they provide an avenue to conjugate any two peptides to the choice of the user and as such are a novel contribution to the BioBrick Registry. Here we describe in detail the design considerations and background information that informed our BioBrick design.

SortaseA Parts

SortaseA (SrtA) from Staphylococcus aureus is a transpeptidase that has been used for protein engineering as it can post-translationally conjugate the N-terminal and C-terminal ends of two proteins. In our project, we used an improved SrtA enzyme [1] that requires two tags on the proteins to be conjugated which we have turned to basic parts:

N-terminal polyglycine (G)_n motif (BBa_K3072500)
C-terminal LPXTG motif (BBa_K3072510)

These basic parts can be introduced to any two peptides of interest and the SrtA-catalyzed conjugation would follow the below scheme:

**Figure 1:** Schematic of SrtA basic BioBricks.

It should be noted that the polyglycine tag needs to be exposed on the protein displaying it. This introduces an issue as the initiating methionine on all peptides will block SrtA action. Thus, the N-terminal tag must be placed downstream a protease recognition sequence or a localization tag, which when cleaved, expose the (G)_n tag on the N-terminus of the mature peptide.

SrtA tagged proteins have been added to the registry before, however, previous teams have not included the tags themselves as basic parts.

M13 Bacteriophage Parts

Using the basic parts described above, we designed parts that also allow the post-translational modification of the p8 M13 bacteriophage coat protein. Modifying p8 has value as it is the main coat protein of M13, thus allows for the most sites available for modification as compared to the other M13 proteins. It has been previously shown by Yoo and Chung that direct expression of the large fusions on the p8 protein hinders the structural integrity of the M13 phage and prevents phage assembly, but introducing the N-terminal SrtA tag allowed for conjugation without hindering the assembly [2].

**Figure 2:** Schematic of M13 coat proteins [3]

This system has not been introduced to the Registry, so our team thought it beneficial to add so that other teams may use the method in the future. One aspect of the p8 gene which is not well documented in the literature is that the p8 protein has a localization tag at its N-terminus which gets cleaved in the mature protein. We discovered this fact after realizing that M13 phage gets assembled at the host membrane and running the p8 gene through a bioinformatic tool that searches for membrane localization sequences.

**Figure 3:** Signal peptide analysis of M13 p8 peptide Output corroborates that a signal peptide leader sequence is present with the cleavage site noted as Alanine 24 (dashed green spike). Generated with SignalP-5.0

This lead to our team creating two additional basic parts:

M13 p8 signal peptide (BBa_K3072520)
M13 p8 Fragment (downstream signal peptide (SP)) (BBa_K3072904)

With these M13 basic parts, we designed a modified p8 gene (BBa_K3072020) that has the N-terminal SrtA tag downstream the M13 SP and upstream the p8 coding sequence. When introduced into the M13 phagemid and transformed into the host with a helper phage, intact M13 phage particles should be produced with the SrtA displayed along the long axis of the assembled phage.

**Figure 4:** Schematic of M13 basic BioBricks.

**Figure 5:** Schematic of M13 p8 Composite BioBrick.

AmajLime Parts

To accompany the SrtA tagged p8 M13 protein, we also designed various BioBricks that can produce visual reporter proteins that display the C-terminal SrtA tag.

T7-promoter regulated amajLime expression construct (BBa_K3072010)
Constitutive promoter amajLime expression construct (BBa_K3072015)
T7-promoter regulated mRFP expression construct (BBa_K3072030)

In our work with the amajLime, we aimed to characterize a previous part BBa_K1033916 which was used as inspiration of the design of our part BBa_K3072010. See Characterization to find out how we further investigated this previously submitted part.

We also aimed to improve a previous part BBa_K3072014 which is amajLime expression construct which uses a constitutive promoter and RBS from the iGEM catalog. See Contribution to find out how our validated contribution to the Registry and improvements to BBa_K3072014 are progressing.

Parts Table

<groupparts>iGEM19 UAlberta</groupparts>

References