In synthetic biology it is of great importance to have well characterized genetic elements. For model organisms, these genetic elements are usually well characterized, however for non-model organisms, like M. extorquens, this toolkit is lacking1, 2. The ability to regulate gene expression provides many options for genetic engineering, therefore, we decided to build a parts collection testing constitutive and inducible promoters. As a backbone, a oriV-traJ’ origin with TcR was used (pTE100, which was a gift from Dr. Schada Von Borzyskowski (Addgene plasmid # 59395))1. The promoter strength was assessed using an mCherry testing cassette. We included the constitutively active promoters PmxaF, PfumC, PcoxB and Ptuf, which were previously characterized in M. extorquens by Schada Von Borzyskowski et al. (2015)1. We included the IPTG inducible promoters PL/O4/A1, which were previously published by Carrillo et al. (2019)2. Additionally, we included an inducible promoter that was not yet characterized for M. extorquens specifically. In 2014, Kaczmarczyk et al. published a vanillate inducible promoter, PV10 for expression in Sphingomonas3.

Constitutive Promoters

Table 1: Overview of the constitutive promoters in the parts collection

Name Part Promoter strength
PfumC BBa_K4721000 Low
PcoxB BBa_K4721001 Low
PmxaF BBa_K4721002 High
Ptuf BBa_K4721003 Medium

Inducible Promoters

Table 2: Overview of the composite inducible promoters in the parts collection

Name Composite Part Composite Part Name Basic Parts Basic Part Basic Part Features
PL/O4/A1 IPTG inducible system BBa_K4721006 PL/O4/A1 BBa_K4721004 Promoter, Operator
lacIq promoter BBa_K4721005 Promoter
lacIq repressor gene BBa_K1222003 Coding
lacIq terminator BBa_K4721007 Terminator
PV10 vanillate inducible system BBa_K4721008 VanR repressor gene BBa_K4721009 Coding
RBS to combine with Pbla-mut1T BBa_K4721013 RBS
Pbla-mut1T BBa_K4721012 Promoter
PV10 BBa_K4721011 RBS, Operator
RBS to combine with PV10 BBa_K4721010 RBS
  1. Schada Von Borzyskowski L, Remus-Emsermann M, Weishaupt R, Vorholt JA, Erb TJ. A set of versatile brick vectors and promoters for the assembly, expression, and integration of synthetic operons in methylobacterium extorquens am1 and other alphaproteobacteria. ACS Synth Biol. 2015;4(4):430-443. doi:10.1021/sb500221v
  2. Carrillo M, Wagner M, Petit F, Dransfeld A, Becker A, Erb TJ. Design and Control of Extrachromosomal Elements in Methylorubrum extorquens AM1. ACS Synth Biol. 2019;8(11):2451-2456. doi:10.1021/acssynbio.9b00220
  3. Kaczmarczyk A, Vorholt JA, Francez-Charlot A. Synthetic vanillate-regulated promoter for graded gene expression in Sphingomonas. Published online 2014. doi:10.1038/srep06453