Team:NTNU Trondheim/Parts


Overview of parts in this project:

Medal Part name Type Description Length (bp)
Bronze BBa_K325909 Generator Lux Operon (under pBAD) 7637
Silver BBa_K2265000 Device MP6 mutator (original) 4908

Our team improved the characterization of the BioBrick BBa_K325909 and designed and characterized the BioBrick BBa_K2265000. Both are documented in the iGEM Registry.

Biobrick contribution: The MP6 mutator (original)

The MP6 mutator (original) biobrick is a plasmid with an operon under control of the promoter pBAD together with the transcription factor gene AraC, and six other genes that disrupt DNA replication: DnaQ926, Dam, SeqA, EmrR, Ugi and Cda1. We designed this biobrick to accelerate bacterial mutation rate, which could be useful for our evolution experiments.

The DnaQ926 is a dominant negative variant of the Escherichia coli DNA pol III proofreading domain. Dam has a strong mutator effect due to impaired mismatch repair. The SeqA protein negatively regulates the initiation of DNA replication at the origin of replication. A low expression of SeqA reduces the transcription of the MP6 plasmid in absence of arabinose. The EmrR gene codes for a protein that compromises intracellular dNTP pools. Ugi codes for a protein that inhibit Ung, a mutagenesis preventing enzyme, through mimicry of structural and electronic features of uracil-containing DNA. CDA1 codes for a cytidine deaminase that is reported to promote the mutation of both prokaryotic and eukaryotic genomic DNA.[1]

The biobrick is based on the MP6 plasmid from addgene (Addgene plasmid # 69669). The original plasmid was changed in order to make it a biobrick. The change done to the plasmid was removing a Spel restriction site by changing an A to a C (nucleotide number 4971 in the original MP6 plasmid). A biobrick prefix was also added in front of the araC gene and a biobrick suffix after the CDA1 gene.

[1] Development of potent in vivo mutagenesis plasmids with broad mutational spectra. Badran AH, Liu DR.  Nat Commun. 2015 Oct 7;6:8425. doi: 10.1038/ncomms9425. 10.1038/ncomms9425 PubMed 26443021

Characterization of the Lux Operon biobrick

We chose to improve this biobrick as we originally wanted to use the lux operon as a reporter for our arabinose-inducible system. In this way, we could test how the expression from the pBAD promoter was dependent on arabinose concentration.

By looking at previous characterisation results of the biobrick, we thought it would work reasonably to use DH5α competent E. coli cells and an incubation temperature of 37 °C. However, when testing with 0.01M arabinose concentrations, no observable luminescence was detected at any time after induction. Therefore, we decided to characterise the biobrick by measuring the luminescence from the lux operon at different arabinose concentrations and temperatures. We measured the luminescence with a Tecan M200 plate reader, using four different temperatures and six different arabinose concentrations. As the machine could only perform heating and not cooling, it was challenging to measure at temperatures below 29 °C.

The results are given on the biobrick parts page.