Here you can find a basic overview of the parts we used in our construct and our various testing plasmids.

In order to test each of the four modules of our construct, we designed specific testing plasmids that allow us to better observe and characterize the function of our parts. We used Golden Gate assembly to build the plasmids, therefore we have various Level 1 and Level 2 plasmids. There is a description and an image of all composite parts in our testing plasmids.

1. Sensing module:

The table below contains all basic parts and testing plasmids we constructed for the sensing component of our circuit. It is composed of a protein that forms a complex with the sugar xylose and binds a specific promoter.

Type of part Name in Part registry RhyzUp name Description Function of part Image of composite part
Basic BBa_K5250000 p01Rhyz01 XutR Protein that binds xylose and forms a complex
Basic BBa_K5250001 p03Rhyz05 Pxut Promoter, induced by the complex between xylose and the XutR protein
Composite level 1 BBa_K5250011 pT11Rhyz01 constitutive promoter, strong RBS, XutR Constitutive expression of XutR BBa_K5250011
Composite Level 1 BBa_K5250012 pT11Rhyz02 Pxut promoter, strong RBS, GFP Expression of GFP induced by the XutR-xylose complex binding to Pxut BBa_K5250012
Composite Level 2 BBa_K5250013 pT12Rhyz01 Constitutive promoter, XutR + Pxut, GFP Sensing testing plasmid: constitutive expression of XutR, when xylose is present XutR-xylose complex binds to Pxut and expresses GFP BBa_K5250013

2. Biofilm overexpression:

Our biofilm overexpression module consists of the following basic and composite parts. On one hand we boost biofilm by inducing the expression of a diguanylate cyclase, on the other hand we knock down a phosphodiesterase using the CRISPRi system.

Type of part Name in Part registry RhyzUp name Description Function of part Image of composite part
Basic BBa_K5250006 p03Rhyz04 DGC P. sp. IsoF wild type + Tag DGC overexpression
Basic BBa_K5250005 p03Rhyz06 DGC P. sp. IsoF R240A + Tag DGC overexpression
Basic BBa_K5250004 p03Rhyz07 DGC P. sp. IsoF R196A + Tag DGC overexpression
Basic BBa_K5250007 p03Rhyz08 DGC WspR R242A + Tag DGC overexpression
Basic BBa_K748003 p03Rhyz09 DGC WspR DGC overexpression
Composite BBa_K5250014 pT21Rhyz01 Rhamnose promoter, strong RBS, P. sp. IsoF DGC wild type, Terminator P. sp. IsoF wild type DGC expressed after a rhamnose promoter BBa_K5250014
Composite BBa_K5250015 pT21Rhyz02 Rhamnose promoter, strong RBS, P. sp. IsoF DGC R196A, Terminator P. sp. IsoF mutated DGC expressed after a rhamnose promoter BBa_K5250015
Composite BBa_K5250016 pT21Rhyz03 Rhamnose promoter, strong RBS, P. sp. IsoF DGC R240A, Terminator P. sp. IsoF mutated DGC expressed after a rhamnose promoter BBa_K5250016
Composite BBa_K5250017 pT21Rhyz04 Rhamnose promoter, strong RBS, P. aeruginosa DGC WspR, Terminator P. aeruginosa wild type DGC expressed after a rhamnose promoter BBa_K5250017
Composite BBa_K5250018 pT21Rhyz05 Rhamnose promoter, strong RBS, P. aeruginosa DGC WspR R242A, Terminator P. aeruginosa mutated DGC expressed after a rhamnose promoter BBa_K5250018
Basic BBa_K5250008 p03Rhyz13 PDE-targeting sgRNA sgRNA to knock down PDE
Composite BBa_K5250019 p1DRhyz04 constitutive promoter, strong RBS, sgRNA, Terminator Expresses the sgRNA needed to knock the PDE down via CRISPRi BBa_K5250019

3. Feedback loop:

We designed a feedback loop which consists of the T7-like RNA polymerase of the Pseudomonas phage Pf-10 and the respective T7-like promoter it binds to. Besides further boosting the biofilm overexpression, the feedback loop also links the separate components of our construct.

Type of part Name in Part registry RhyzUp name Description Function of part Image of composite part
Basic BBa_K5250002 p03Rhyz10 T7-like RNA polymerase (T7-like RNAP) Binds to the T7-like promoter
Basic BBa_K5250003 p01Rhyz03 T7-like promoter Recruits the T7 RNA polymerase, which triggers transcription
Composite level 1 testing plasmid BBa_K5250020 pT31Rhyz01 Constitutive promoter, RBS, T7-like RNAP, Terminator Expresses T7-like RNAP constitutively BBa_K5250020
Composite Level 1 testing plasmid BBa_K5250021 pT31Rhyz02 T7-like promoter, RBS, GFP, Terminator GFP expressed after activation of the T7-like promoter BBa_K5250021
Composite Level 1 testing plasmid BBa_K5250022 pT31Rhyz03 T7-like promoter, RBS, RFP, Terminator, on 1C backbone Dummy gene, needed for golden Gate assembly BBa_K5250022
Composite Level 1 testing plasmid BBa_K5250023 pT31Rhyz04 T7-like promoter, RBS, T7-like RNAP, Terminator T7-like promoter can activate expression of more T7-like RNAP after induced by T7-like RNAP BBa_K5250023
Composite Level 1 testing plasmid BBa_K5250022 pT31Rhyz06 T7-like promoter, RBS, RFP, Terminator, on 1D backbone Dummy gene, needed for golden Gate assembly BBa_K5250022
Composite Level 2 testing plasmid BBa_K5250024 pT32Rhyz01 Constitutive promoter, strong RBS, T7-like RNAP + T7-like promoter, strong RBS, GFP + 2x dummy genes Constitutive expression of T7-like RNAP, activates one T7-like promoter expressing GFP BBa_K5250024
Composite Level 2 testing plasmid BBa_K5250025 pT32Rhyz02 Constitutive promoter, RBS, RNAP + T7-like promoter, RBS, dummy gene + T7-like promoter, RBS, GFP + dummy gene Constitutive expression of T7-like RNAP, activates two T7-like promoters, one of them is expressing GFP BBa_K5250025
Composite Level 2 testing plasmid BBa_K5250026 pT32Rhyz03 Constitutive promoter, RBS, T7-like RNAP + T7-like promoter, RBS, T7-like RNAP + T7-like promoter, RBS, GFP + dummy gene Constitutive expression of T7-like RNAP, activates two T7-like promoters, both expresses GFP BBa_K5250026

4. Kill switch:

Our kill switch consists of a DNA gyrase inhibiting toxin and its respective antitoxin. The toxin is expressed after the T7-like promoter, while the antitoxin - after the xylose promoter.

The transcriptional unit expresses a toxin which is induced by a rhamnose promoter and a weak RBS.

Type of part Name in Part registry RhyzUp name Description Function of part Image of composite part
Basic BBa_K3512001 p03Rhyz02 Toxin Inhibits DNA gyrase
Basic BBa_K3512002 p03Rhyz01 Antitoxin Counteracts the gyrase-inhibiting toxin
Composite testing plasmid BBa_K5250027 pT41Rhyz01 rhamnose promoter, weak RBS, toxin Toxin induced by a rhamnose promoter and a weak RBS BBa_K5250027
Composite testing plasmid BBa_K5250028 pT41Rhyz02 rhamnose promoter, medium RBS, toxin Toxin induced by a rhamnose promoter and a medium strong RBS BBa_K5250028
Composite testing plasmid BBa_K5250029 pT41Rhyz03 rhamnose promoter, strong RBS, toxin Toxin induced by a rhamnose promoter and a strong RBS BBa_K5250029
Composite testing plasmid BBa_K5250030 pT41Rhyz04 rhamnose promoter, strong RBS, antitoxin Antitoxin induced by a rhamnose promoter BBa_K5250030
Composite testing Plasmid Level 2 BBa_K5250031 pT42Rhyz01 Rhamnose promoter, strong. RBS, toxin + Rhamnose promoter, strong RBS, antitoxin A Level 2 testing plasmid that expressed both the CcdB toxin and CcdA antitoxin after a rhamnose promoter BBa_K5250031

Final construct parts

After we conducted the necessary experiments and chose the best-performing parts, we used Golden Gate to assemble our final construct. It consists of 2 Level 2 plasmids, each of them with 4 transcriptional units. For the biofilm overexpression module we chose the P. sp. IsoF R196A mutation after we conducted statistical analysis for our c-di-GMP assays. Additionally, we selected the strongest RBS for our kill switch.

  1. pT7-like + DGC WspR R242A, Pxut + T7-like RNAP, pT7-like + T7-like RNAP, pT7-like + PDE-inhibiting sgRNA
  2. pT7-like + DGC WspR R242A, constitutive promoter + XutR, pT7-like + toxin, Pxut + antitoxin

Type of part Name in Part registry RhyzUp name TU1 TU2 TU3 TU4
Composite Level 2 plasmid BBa_K5250009 p21Rhyz02 p1ARhyz05 p1BRhyz01 p1CRhyz01 p1DRhyz05
Composite Level 2 plasmid BBa_K5250010 p22Rhyz02 p1ARhyz05 p1BRhyz02 p1CRhyz02 p1DRhyz01