Difference between revisions of "Part:BBa K2265000"

Latest revision as of 21:36, 1 November 2017

MP6 mutator (original)
The biobrick is a plasmid with one 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.

Usage and Biology

The DnaQ926 is a dominant negative variant of the E. 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.

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.

Characterization

To test its mutagenic activity after inducer and repressor addition, we used a reversion assay based on the plasmid pLA230 (from Addgene). The pLA230 plasmid contains a non-functional Ampicillin resistance gene. The inactivity of the gene is due to a point mutation, and a reversion of this mutation will lead to Ampicillin resistance in a cell containing the plasmid.

In order to characterize the mutation rate of the MP6 biobrick, it was transformed together with pLA230 into E.coli DH5α. The doubly transformed cells were grown with either glucose or arabinose (see protocol) and incubated in 37ºC overnight. The cells were then plated on agar containing Ampicillin. The number of colonies formed was an indication of the mutation activity of MP6, as only bacteria containing pLA230 where the point mutation was reverted would survive on the Amp containing agar plates.

Reversion frequency of MP6 biobrick induced with arabinose or repressed with glucose.

The reversion frequency was quantified by dividing the number of colonies surviving on ampicillin by the number of colonies growing without ampicillin (scaling with the dilution factors accordingly). We performed two testing rounds on our MP6 biobrick, giving on average a reversion frequency of 10-5 when induced and 10-6 when repressed. In order to compare our biobrick with the original MP6 we quantified the relative reversion frequencies for both, that is how many times more revertants we got when the plasmid was induced than when it was not. We got ratios of 19 and 21 respectively. Thus the two plasmids performed quite similarly.

Relative reversion frequencies of our MP6 biobrick and orginal MP6.

Reversion protocol

1) Doubletransform E.coli with MP6 and pla230 (Addgene reversion assay plasmid) and let it incubate for 18 hours exactly.

2) Split the culture and add glucose to a final concentration of 20 mM to one subculture, the same concentration of arabinose to the other.

3) Incubate for 24 hours.

4) Plate on LA+Amp+glucose and LA+glucose plates in 10x dilutions.

5) Count after the colonies have grown sufficiently.

  Sequence and Features