Registry Parts

Here you can find the biological parts and composite parts we have used in the project.

Parts Description

Our project has been about designing, testing, and expressing DARPins specific to our project. We have been working with two different DARPins, one that is a well-known DARPin that binds to GFP, and a DARPin that would bind to the signalling molecule AIP. Our collection is made up of the parts needed to test and express these DARPins. More information about the background of the projects can be found here. To read more about how we have used the parts, go to the protocols and lab book page.

Basic Parts

Below is a list of the Aalto-Helsinki basic parts that we used for our composite parts.

Part Type Description Length (bp)
BBa_K4159002 T7 T7 promoter with stem-loop 32 bp
BBa_K4159001 RBS RBS sequence 23 bp
BBa_K4159003 Spacer Spacer sequence for ribosome display of the DARPin construct 217 bp
BBa_K4159004 Tag 6xHis-tag 36 bp
BBa_K4159005 CDS GFP binding DARPin 483 bp
BBa_K4159006 Tag Avi-tag 45 bp
BBa_K4159007 CDS AgrC encoding gene 1290 bp
BBa_K4159008 CDS AgrA encoding gene 627 bp
BBa_K4159010 Regulatory Reversed P2 promoter 96 bp
BBa_K4159010 CDS AIP binding DARPin 471 bp

Composite Parts

The composite parts consist of parts we have used both in the bioreporter and in the ribosome display.

Part Type Description Length (bp)
BBa_K4159009 CDS Coding AgrC + AgrA. AgrC receptor and AgrA signalling protein from S. epidermidis 2117 bp
BBa_K4159000 CDS Coding GFP-DARPin. GFP binding DARPin with T7 promoter and spacer 791 bp
BBa_K4159011 Regulatory P2 promoter and pBAD promoter sequence in opposite directions, with RBS. 256 bp
BBa_K4159012 CDS Composite part, that expresses the GFP DARPin 618 bp

Below is a brief description of the design of our plasmid constructs we have used for our basic and composite parts. Figure 1. shows the plasmid we used firstly to express the GFP protein that we needed for our ribosome display. We also cut the plasmid at three different sites for the bioreporter introduction (see Figure 2). BBa_K4159011 was digested and inserted at the NcoI and XbaI restriction sites, and BBa_K4159009 was digested and inserted at the XbaI and PaeI(SphI) restriction sites.

Circular overview of pET28a-sfGFP plasmid
Figure 1. Overview of pET28a-sfGFP plasmid used as backbone for bioreporter construct (Addgene)
Construct visualization of the bioreporter
Figure 2. Bioreporter construct
Circular overview of pET-His6x-rAPOBEC1-XTEN-nCas9-UGI-NSL expression plasmid
Figure 3. pET-His6x-rAPOBEC1-XTEN-nCas9-UGI-NSL expression plasmid used for GFP-DARPin expression

For our GFP-binding DARPin expression we got the pET-His6x-rAPOBEC1-XTEN-nCas9-UGI-NSL expression plasmid from our mentor Sami Jalil. We inserted the BBa_K4159000 at the XbaI and XhoI restriction sites.