Part:BBa_K729004:Experience

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Testing the compatibility of the nuclease with Azo-dyes

The BBa_K729004 periplasmic nuclease that UCL iGEM 2012 submitted and characterised was able to demonstrate positive DNase agar assays. This assay involves washing DNA-containing agar with HCl after streaking bacteria. After this wash, a ‘halo’ surrounding the streaked bacteria indicates that extracellularly secreted DNase digested the DNA surrounding the colonies within the agar. In order to further characterise this BioBrick and incorporate it into our project as a biosafety method of minimising the transfer of extracellular DNA, we decided to test whether BBa_K729004 would function in the presence of Azo-Dyes.



Figure 1 - BBa_K729004 BBa_K729004 periplasmic nuclease enzyme shows functionality in the presence of multiple Azo-dyes. Figure showing that the BBa_K729004 periplasmic nuclease enzyme is still able to digest the surrounding DNA in the DNase agar. Figure 1a and 1b demonstrate the presence of halos around colonies on plates with and without Acid Orange 7 (AO7) azo-dye. Figure 1c and 1d demonstrate the presence of halos around colonies on plates with and without Reactive Black 5 (RB5). All azo-dye agar plates were made with a 1:500 dilution of 0.5mgml-1 dye.

Since the azo-dye degradation would be taking place in industrial environments, being the products of the bioreaction at one point dumped into other channels of water-treatment or into the environment, it is essential that there are certain barriers that stop the DNA from our genetically modified organisms from potentially being transferred into wild-type strains. On a basic first-level defense, this assay suggests that horizontal gene transfer could be inhibited in an azo-dye contaminated environment byBBa_K729004 , as it has proven effective in degrading extracellular bacterial DNA.

By UCL 2014

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Using BBa_K729004 to degrade biofilms, by [http://2015.igem.org/Team:Oxford Team Oxford 2015]

We ordered this part from the Registry to test if Staphylococcal nuclease is capable of degrading E. coli biofilms. We received the part on 23rd July 2015 from iGEM HQ.

Despite the lack of annotations on the part sequence, this part actually consists of the nucleotide sequence for the DsbA 2-19 signal sequence from nucleotide 4 to nucleotide 57, followed by the sequence for Staphylococcal nuclease from nucleotide 58 to 558. In view of that, we were interested in 2 aspects of this part:

1. Whether the DsbA 2-19 sequence is able to facilitate the export of this part of expression host organism E. coli MG1655

2. Whether the Staphylococcal nuclease can degrade E. coli biofilms (it was shown to degrade S. aureus biofilms in [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005822 Mann et al, 2009 {PLoS ONE 4(6))])

We used PCR to add on a BspHI site upstream of the coding sequence, and subsequently ligated it into the commercial expression vector pBAD/HisB so that we can use L-arabinose to induce the expression of this gene.

We cloned the BBa_K729004[pBAD] plasmid into E. coli MG1655 and using arabinose induction showed that:

- The staphylococcal nuclease encoded by this part is successfully secreted into the extracellular media

We added 0.2% L-arabinose to a subculture of ''E. coli'' MG1655 BBa_K729004[pBAD] at OD₆₀₀ = ~0.8, and after 4 hours of secretion we isolated the supernatant and purified the protein content by TCA precipitation. We dissolved the precipitate in SDS and loaded it into Lane B in the image above. A ~21 kDa band corresponding to the size of DsbA-DNase can be seen. Lane A is the control lane whereby a separate subculture of the same cells which had no arabinose added into it was similarly treated with TCA and SDS.

- The staphylococcal nuclease encoded by this part can degrade E. coli biofilms

We added 0.2% L-arabinose to a 1:100 subculture of ''E. coli'' MG1655 BBa_K729004[pBAD] in a 96-well plate and incubated it at room temperature for 3 days, after which we washed off the planktonic cells in the culture through gentle rinsing and stained the adherent biofilm layer using 0.1% crystal violet solution. The staining results, when measured at OD₅₉₀, showed reduced biofilm formation by the cells whose nuclease gene was expressed (the leftmost column in the image above), when compared to the same cells whose nuclease gene was not expressed (the middle column) and ''E. coli'' MG1655 with only a blank pBAD/HisB plasmid in them.

In conclusion, we have both improved the characterization as well as increased the functionality of this part.

As a follow up, we have also created a new part based on this part, BBa_K1659301 which has an additional hexahistidine tag downstream of the nuclease gene to allow metal-affinity purification of the protein. Please visit the linked page to see the characterization of our part.

By Team Oxford 2015

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