Abstract
This page lists the parts that our team created and used during our project. We are proud to introduce 8 new basic parts and 5 new composite parts to the synthetic biology world. These bricks serve different purposes from increasing fluoride resistance to detecting fluoride ions in a solution and most importantly, accelerated directed evolution through orthogonal replication in Escherichia coli. We hope that they will help iGEMers and scientists worldwide advance science.
Introduction
All biobricks were designed by our team using benchling. Most of them were ordered in fragments to IDT or Twist Bioscience, which allowed for codon optimization of specific genes for E. coli. In the case of fluC, its CDS was amplified from Pseudomonas putida KT2440 genome using PCR amplification. The team then assembled all DNA fragments using Gibson assembly in their respective vectors.
Composite parts
| Type | Name | Link | Purpose | Representation | Importance in PFAway | Length (bp) | Status | RFC |
|---|---|---|---|---|---|---|---|---|
| Composite | Synthetic replication operon | BBa_25HKSDJN | Composite part encoding, in the form of an operon, the necessary PRD1 proteins to maintain and mutate the linear replicon. Inducible with IPTG. |  |
*** | 3 680 | Successfully assembled in the form of a linear DNA fragment. | 10 |
| Composite | Linear replicon | BBa_25794BPU | Composite part allowing for the orthogonal replication and fast evolution of the Haloacid Dehalogenase type II. |  |
*** | 2 214 | Successfully cloned. | 10 & 1 000 |
| Composite | FluorMango expression cassette | BBa_25IYP52O | Composite part for in vitro expression of the FluorMango RNA using T7 RNA polymerase. |  |
*** | 95 | Successfully constructed and characterized. | 10 & 1 000 |
| Composite | GFP fluoride biosensor | BBa_2527DQMU | This part allows for the expression of a GFP in the presence of fluoride. |  |
* | 1 071 | Successfully cloned. Did not lead to the expected results. | 10 & 1 000 |
| Composite | Empty linear replicon | BBa_25P82DIA | Composite part allowing for the orthogonal replication and fast evolution of a user-defined gene. |  |
** | 1 553 | Successfully cloned. | 10 & 1 000 |
Basic parts
| Type | Name | Link | Purpose | Representation | Importance in PFAway | Length (bp) | Status | RFC |
|---|---|---|---|---|---|---|---|---|
| Basic | fluC | BBa_25321TIX | Fluoride-specific ion channel in Pseudomonas putida. Overexpressed to increase fluoride resistance. |  |
*** | 375 | Successfully cloned and characterized. | 10 & 1 000 |
| Basic | Ptac | BBa_K4595010 | Ptac promoter is a fusion of Ptrp and Plac.Ptacis IPTG inducible. |  |
* | 29 | Successfully assembled in BBa_25HKSDJN, BBa_25794BPU and BBa_25P82DIA. | 10 & 1 000 |
| Basic | lacO | BBa_K1624002 | lac> operator. Regulates downstream gene expression, allowing transcription upon IPTG induction. |  |
* | 23 | Successfully assembled in BBa_25HKSDJN | 10 & 1 000 |
| Basic | Terminal Protein | BBa_25GVXNT9 | Terminal protein of phage PRD1. Binds to ITRs and enables replication by the DNA polymerase. Optimized for gene expression in E. coli. |  |
* | 780 | Successfully assembled in BBa_25HKSDJN | 10 |
| Basic | DNA polymerase | BBa_25AOWIG9 | DNA polymerase of phage PRD1. Uses ITR/terminal protein complex to initiate replication. Has two point mutations aiming at increasing its mutation rate. Optimized for gene expression in E.coli. |  |
** | 1 662 | Successfully assembled in BBa_25HKSDJN | 10 |
| Basic | ssDNA binding protein | BBa_25XMK7N9 | ssDNA binding protein of phage PRD1. Stabilizes replication. |  |
* | 483 | Successfully assembled in BBa_25HKSDJN | 10 & 1 000 |
| Basic | dsDNA binding protein | BBa_251EDGZD | dsDNA binding protein of phage PRD1. Stabilizes replication. Not essential for ORep. |  |
* | 285 | Successfully assembled in BBa_25HKSDJN | 10 & 1 000 |
| Basic | ITR | BBa_253ZBG0C | Minimal Inverted Terminal Repeats of phage PRD1 genome. Act as the origin of replication. |  |
* | 18 | Successfully assembled in BBa_25794BPU and BBa_25P82DIA. | 10 & 1 000 |
| Basic | KanR | BBa_K5042230 | Kanamycin resistance gene. Serves as a selective marker. |  |
* | 816 | Successfully assembled in BBa_25794BPU and BBa_25P82DIA. | 10 & 1 000 |
| Basic | dehH2 | BBa_2584F6AG | Haloacid Dehalogenase type II. Candidate for performing PFAS degradation. Optimized for gene expression in E. coli. |  |
** | 675 | Successfully assembled in BBa_25794BPU | 10 & 1 000 |
| Basic | T7 terminator | BBa_K731721 | Transcriptional terminator from T7 phage. |  |
* | 48 | Successfully assembled in BBa_25794BPU, BBa_25P82DIA and BBa_2527DQMU. | 10 & 1 000 |
| Basic | PT7 | BBa_R0085 | Promoter from T7 phage. |  |
* | 23 | Successfully assembled in BBa_25IYP52O | 10 & 1 000 |
| Basic | FluorMango coding sequence | BBa_25K2IGV0 | dsDNA encoding a fluoride sensitive RNA biosensor. Becomes fluorescent when bound to fluoride and TO1-3PEG-biotin. |  |
* | 70 | Successfully assembled in BBa_25IYP52O | 10 & 1 000 |
| Basic | PJ23119 | BBa_K5303058 | PJ23119 is the consensus and strongest promoter of the PAnderson family of constitutive promoters in E. coli. |  |
* | 35 | Successfully assembled in BBa_2527DQMU | 10 & 1 000 |
| Basic | Fluoride riboswitch | BBa_K4868000 | Fluoride-dependent riboswitch. Allows for translation of downstream genes upon fluoride binding. |  |
* | 85 | Successfully assembled in BBa_2527DQMU | 10 & 1 000 |
| Basic | sfGFP | BBa_K2541400 | Superfolder green fluorescent protein. |  |
* | 720 | Successfully assembled in BBa_2527DQMU | 10 & 1 000 |
| Basic | T1 terminator | BBa_B0010 | Transcriptional terminator from rrnB gene of E. coli. |  |
* | 80 | Successfully assembled in BBa_2527DQMU | 10 & 1 000 |