Parts

Part 1 Calculating

Introduction

This subcollection contains the components used to build the calculator, including high-pass, band-pass and connector.

These components involve mutually orthogonal AHL bacterial quorum sensing systems such as Las, Rhl, Tra, and Cin, thereby forming three types of logic gates: AND, OR, and XOR, and creating two circuits: high-pass and band-pass. They are connected through the connector, ultimately achieving addition operations.

We hope these tools can inspire other iGEM teams and synthetic biology researchers in bacterial quorum sensing systems and biological computing.

Name Description Type Main Functions
BBa_25EK8LOU CinR Basic The CinR protein is a key protein in bacterial quorum sensing. It is activated by the bacterial quorum sensing molecule 3-OH-C14:1 HSL and then activates the Pcin promoter.
BBa_25J6IAUX VqmA Basic The VqmA protein is a key protein involved in bacterial quorum sensing. It is activated by the bacterial quorum sensing molecule DPO and subsequently activates the Pvqm promoter.
BBa_25B8ZS98 Pvqm Basic Pvqm is a promoter activated by the VqmA protein. In our project, Pvqm is placed upstream of the GFP gene, so that the GFP fluorescence intensity can reflect the concentration of DPO in the environment.
BBa_25FFR1B9 TraR Basic The TraR protein is a key protein in bacterial quorum sensing. It is activated by the bacterial quorum sensing molecule 3-oxo-C8 HSL and subsequently activates the Ptra promoter.
BBa_25EEWCIY Ptra Basic Ptra is a promoter activated by the TraR protein. In our project, Ptra is placed upstream of the GFP gene, so that the GFP fluorescence intensity can reflect the concentration of TraR in the environment.
BBa_25WIM9S3 TraI Basic TraI gene encodes a key enzyme in the bacterial quorum sensing system, whose main function is to synthesize the signaling molecule 3-oxo-C8 HSL.
BBa_25B3PA10 CinI Basic CinI gene encodes a key enzyme in the bacterial quorum sensing system, whose main function is to synthesize the signaling molecule 3-OH-C14:1 HSL.
BBa_25JQHE92 RhlI Basic The Rhl acyl-homoserine-lactone synthase that has undergone codon optimization at three sites encodes a key enzyme in the bacterial quorum sensing system. Its main function is to synthesize the signaling molecule C4-HSL.
BBa_255WKJW5 Prhl-phlO Basic An rhl promoter with one phlO sequence upstream and four phlO downstream, so that it will be repressed by phlF.
BBa_25X31R3Y PT7-phlO4 Basic A T7 promoter core with one phlO sequence upstream and four phlO downstream, so that it will be repressed by phlF strongly.
BBa_253EJSXL VqmA expression system Composite It is used for the expression of the VqmA protein.
BBa_253D9E4D RhlR-CinI Composite The RhlR-CinI composite element is the RhlR gene with the CinI gene regulated by the Prhl promoter, forming a typical quorum sensing regulatory circuit. Prhl is a promoter specifically regulated by the RhlR protein.
BBa_25Q9W55S RhlR-LasI Composite The RhlR-LasI composite element is the RhlR gene with the LasI gene regulated by the Prhl promoter, forming a typical quorum sensing regulatory circuit. LasI synthase encoded by the lasI gene catalyzes the synthesis of 3OC12-HSL.
BBa_257JONMN RhlR-TraI Composite The RhlR-TraI composite element is the RhlR gene with the TraI gene regulated by the Prhl promoter, forming a typical quorum sensing regulatory circuit. TraI synthase encoded by the TraI gene catalyzes the synthesis of 3-oxo-C8-HSL.
BBa_25GC0MFR Prhl-RBS-PhlF-rrnB T1/T7Te double terminator Composite Express PhlF induced by C4-HSL molecules.
BBa_257SP0LR PJ23106-RBS-RhlR-rrnB T1 terminator Composite Express the C4-HSL receptor protein RhlR continuously.
BBa_259LTFQR PJ23106-RBS-LasR-rrnB T1 terminator Composite Express the 3OC12-HSL receptor protein LasR continuously.
BBa_25LYTXIF Plas-RBS-PhlF-rrnB T1/T7Te double terminator Composite Express PhlF induced by 3OC12-HSL molecules.
BBa_25I8QTAG Plas-RBS-T7 RNAP-rrnB T1 terminator Composite Transcribing T7 RNA polymerase when induced by 3OC12-HSL molecules, which will promote other genes promoted by T7 promoters.
BBa_25PZQXTW Prhl-RBS-T7 RNAP-rrnB T1 terminator Composite Transcribing T7 RNA polymerase when induced by C4-HSL molecules, which will promote other genes promoted by T7 promoters.
BBa_25Q02ZYN Prhl-phlO-sfGFP-lambda t0 terminator Composite This part is a reporter part, in which the sfGFP will be expressed only when the induction of C4-HSL-bind RhlR is stronger than the inhibition of PhlF, acting as a bandpass filter on C4-HSL when PhlF is also induced by C4-HSL.
BBa_25LDZT4A PT7-phlO4-sfGFP-lambda t0 terminator Composite This part is a reporter part, in which the sfGFP will be expressed only when the induction of T7RNAP is stronger than the inhibition of PhlF, acting as a bandpass filter on IPTG when PhlF is controlled by a lac promoter.
BBa_258AVNFT PlacUV5-lac operator-RBS-PhlF-rrnB T1/T7Te double terminator Composite We place the PhlF gene (BBa_K5276009) downstream the Lac UV5 and lac operater to enable its expression intensity to be controlled by IPTG concentration.
BBa_25GV7X6B PT7-phlO-sfGFP-lambda t0 terminator Composite This part initiates sfGFP transcription by T7 promoter connected to a phlO sequence, so that the cells only emit green fluorescence when induced by IPTG and with low PhlF.

Part 2 Refreshing

Introduction

This subcollection contains components for resetting the calculator's state and enabling repeatable calculations, including modules for opto-degradation of AHL molecules and fluorescent proteins.

For AHL molecules, we employed AHL molecules degradation enzymes AiiA. For the fluorescent proteins, we add a protein degradation tag based on AsLOV2, enabling light-inducible degradation of fluorescence proteins.

We hope these tools can inspire other iGEM teams and synthetic biology researchers in opto-degradation.

Name Description Type Main Functions
BBa_25ZEW01P AsLOV2*(543) LOVdeg Basic A modular protein tag based on the AsLOV2 protein that is conditionally degraded in response to blue light in E. coli.
BBa_2508ZG2U mCherry without stop codon Basic This is a mCherry report gene without stop codon, so it can link with other protein tags.
BBa_25MWMLSO AiiA Basic An enzyme that can inactivate the acylhomoserine lactone quorum-sensing signal
BBa_25CWCL0O PlacUV5-lac operator-mCherry-LOVdeg Composite To test the degradation effect of C-terminal AsLOV2*(543) LOVdeg tag in E. coli in response to blue light, we constructed a composite part where we used an IPTG-inducible promoter PlacUV5-lac operator to control the expression of red fluorescence protein mCherry translationally fused to LOVdeg tag. A five amino acid GS linker of 'S-S-G-S-G' was used between mCherry and the LOVdeg tag.