Part:BBa_K1769009
T7+RBS+Lm-def
Usage and Biology
Most fungicide used to cure or prevent potato late blight contain heavy metal, such as copper. these heavy metal will not only kill p.infestans, a pathogen that causes late blight, but also do harm to the plant and environment. Therefore, we created a defensin called lm-def. It is a antifungal peptide from Lepidium meyenii, an Andean crop, that can effectively and specifically inhibit the growth of p.infestans zoospores and destroy the hyphae of p.infestans.
Antimicrobial assay
To test if zoospores and hyphae of p.infestans will be destroyed by our defensin lm-def. We carry out an antimicrobial assay experiment. we incubated in advance the p.infestans on rye agar plates at 18 degrees Celsius for ten days to acquire sporangia and hyphal pieces. Dilution broth solution was then prepared by boiling fresh pea seeds for 20 minutes in distilled water and autoclaved for sterilization. We harvested P. infestans in sterile water; later, we used hemocytometer to count the number of sporangia and diluted it to 250 sporangia per 100 microliter with the dilution broth solution. We took the 100 ul spore suspension and added to it 50 ul of recombinant protein in a 96 well microtiter plate. We used PBS as blank, the pET29b empty vector as protein control, and the spore suspension for comparison. The mixture will then be incubated in darkness at 18 degrees Celsius for an entire day. Spore germination will be measured with a plate reader at 595 nm at the beginning of the test and 24 hours after inoculation. Apart from that, we monitored changes in hyphal growth by physically cutting out agar and placing it in a 12-well tissue culture plate with each well loaded with 1600 ul of lysate while using dilution broth solution as positive control, as it theoretically did not contain any substance that would suppress the growth of the oomycete. We inspected the samples sliced out of the agar every two hours under a light microscope.
Results
The effect of lm-def on E.coli BL21
We want to make sure that lm-def won't do harm to E.coli BL21 so that it won't kill E.coli when E.coli produces the defensin. We added IPTG to induce the production of lm-def when the O.D.600 is 0.6 and measure the O.D.600 of ecoli every 30 minutes.
As we can see in this picture, the growth of E.coli BL21 is not affected by lm-def produced by itself. Therefore, the amount of E.coli won't decrease when producing lm-def
The effect of lm-def on P.infestans
We observe the morphology of P.infestans under microscopy every 2 hours after treating zoospores with lm-def. The picture bellow shows the differences between the morphology begore and after treatment.
Hyphae of p.infestans before treatment
Hyphae of p.infestans after treatment
As we can see on the photo above. The hyphea of the p.infestans in presence of Lm-Def exhibited an unhealthy state at where extensive septum formation and thinning of the unhealthy hyphae could be observed
>Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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