Team:ETH Zurich/Parts

"What I cannot create I do not understand."
- Richard Feynmann

Parts

Many parts were required to complete our project. Some of these parts where used from the Registry, others were designed and cloned for this project. In the following section, we are presenting all parts we used, designed and characterized.

Overview of our genetic design.

BBa_K1847010. Hybrid promoter that is responsive to both IPTG and lactate.

BBa_K2847008. Promoter that shows an improved performance when compared to the natural PlldR

Our part collection

We designed a promoter library based on the wild-type PlldR (BBa_K822000). You can check our results using this promoters here.

Regulatory system design

Content

BioBrick
Promoter designed keeping the original architecture and changing the wild-type promoter by a strong Anderson promoter (BBa_J23100) BBa_K1847007
Promoter designed keeping the original architecture and changing the wild-type promoter by a weak Anderson promoter (BBa_J23117) BBa_K1847008
Promoter designed keeping the original architecture and changing the wild-type promoter by a medium Anderson promoter (BBa_J23118) BBa_K1847009
Promoter designed substituting the promoter by a non-functional DNA sequence, with an Anderson promoter placed in front of the operators (BBa_J23117) BBa_K1847005
Promoter designed substituting the promoter by a non-functional DNA sequence, with an Anderson promoter placed in front of the operators (BBa_J23118) BBa_K1847006
In this design, the spacing between the two operator sites was removed, and the Anderson promoter was placed in front of the operators (BBa_J23100) BBa_K1847002
In this design, the spacing between the two operator sites was removed, and the Anderson promoter was placed in front of the operators (BBa_J23117) BBa_K1847003
In this design, the spacing between the two operator sites was removed, and the Anderson promoter was placed in front of the operators (BBa_J23118) BBa_K1847004
The original PlldR was substituted by Plac and lacO. BBa_K1847010
The original PlldR was substituted by PlacUV5 and lacO. BBa_K1847011
The original PlldR was substituted by a spacer and Plac and lacO were located in front of the promoter. BBa_K1847012
Plac and lacO BBa_K1847013
PlacUV5 and lacO BBa_K1847014

Our new basic parts

In this section we present LldR and LldP, the regulator protein and transporter protein of the LldPRD operon, respectively, as well as a synthetically designed hybrid promoter responsive to lactate and IPTG.

Part

Content

Registry number
LldR Sequence encoding the regulator protein LldR. BBa_K1847001
LldP-LldR Sequence encoding the L-lactate permease LldP and the regulator protein LldR. BBa_K1847016
Synthetically designed hybrid promoter Synthetic promoter inhibited by lactate and IPTG. BBa_K1847010

Our new composite parts

Our composite parts are from used in binding system to E. coli. More information about our experiments on bacterial binding to mammalian cells can be found here.

Part

Content

Registry number
Annexin V Promoter J23114-RBS B0034- Annexin V optimized for Escherichia coli BBa_K1847000
INP-Annexin V Promoter J23114-RBS B0034- INP-Annexin V fusion protein BBa_K1847015

Newly characterized parts of the Registry

Here we present the parts from the Registry where we improved the existing characterization.

Part Name

Description

Registry Number
aiiA Autoinducer inactivation enzyme aiiA (enzyme that inactivates the acylhomoserine lactone (AHL) quorum-sensing signal). Check out our characterization for aiiA! BBa_C0160
pLldR lldPRD operon promoter + RBS from E. coli. In the absence of L-lactate, lldR binds to two operator sites O1 and O2 in the promoter region and inhibits expression. Check out our characterization for pLldR! We also improved the existing promoter by removing the ArcA site and obtained an increased ON/OFF ratio by changing the promoter strength. BBa_K822000

Used parts from of the Registry

In this section we present other parts from the Registry that we used during our project.

Part Name

Description

Registry Number
INP-EYFP Fusion of Ice Nucleation Protein (INP) and Enhanced Yellow Fluorescent Protein (EYFP). Check out our use of INP! BBa_K523013
lacI + LVA lacI repressor from E. coli (+LVA) BBa_C0012
pLuxR Promoter activated by the LuxR protein complexed with the autoinducer homoserine lactone (HSL) BBa_R0062
cI cI repressor from E. coli phage lambda modified with an LVA tail for rapid degradation of the protein BBa_C0051
HylA HlyA-tag+Secretion system, allows a protein to be secreted by means of the alpha-hemolysin secretion system in E. coli BBa_K1166002
InterLab 1 strong promoter Anderson promoter J23101 from the Anderson collection BBa_K823005 (BBa_J23101)
InterLab 2 medium-strong promoter Anderson promoter J23106 from the Anderson collection BBa_K823008 (BBa_J23106)
terminator Transcription terminator for the E. coli RNA polymerase BBa_B0012
double terminator Double terminator consisting of BBa_B0010 and BBa_B0012 BBa_B0015
very strong promoter Strong member of the family of promoters J23100 through J23119 BBa_J23100
gfp (InterLab) intermediate in screening plasmid construction containing GFP BBa_I13504
medium promoter Medium member of the family of promoters J23100 through J23119 BBa_J23118
LuxI autoinducer synthetase for acylhomoserine lactone (AHL), no LVA BBa_C0161
InterLab 3 weak promoter Anderson promoter J23117 from the Anderson collection BBa_K823013 (BBa_J23117)
promoter medium-weak Medium-weak member of the family of promoters J23100 through J23119 BBa_J23114
RBS RBS.3 (medium) derivative of BBa_0030 BBa_B0032
promoter plus GFP (InterLab control) J23151 inserted in the Promoter MeasKit BBa_I20270
terminator Artificial terminator, estimated %T~>90% BBa_B1006

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