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.

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

Additionally, to test the function of defensin against P. infestans in vivo, we sprayed the mixture of the spore suspension and the cell lysate onto the leaves of tomatoes and potatoes after inoculating these lm-def. We compared those treated with defensin with the ones sprayed with only spore suspension.

After one week, we discover dark lesions on the leaves of the plant and observed the tissue under the light microscope. We discover that the infection was more severe on the plant treated with lower concentration of defensin, and that the area of the lesion is smaller on the leaves of the plant treated with higher concentration of defensin. Even though we cannot fully eradicate the infestation of the oomycete, this proved that our product worked to certain degree and can effectively guard the potatoes against the pathogen. In the future we would work further on purifying the product to optimize its function and for real life applications.

Healthy leaves on the plant traeted with lm-def.

Large area of lesions on leaves without treating the plant with lm-def

>Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]