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.
Below is a list of the Aalto-Helsinki basic parts that we used for our composite parts.
|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_K4159005||CDS||GFP binding DARPin||483 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|
The composite parts consist of parts we have used both in the bioreporter and in the ribosome display.
|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.
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.