Team:USP-Brazil/Demonstrate

Demonstrate


We expressed potent effector and characterized two different metabolite sensors to detect a pathogen (dtxR and PtetR_lldR). To complete our goal, finally, we wanted to see if our chassis was able to colonize the mosquito gut so that our project can work in real life!

We were lucky that Prof. Margareth Capurro offered her lab which is a top certified biocontainment level 2 laboratory with an insectary for mosquito cultivation. Helena and Rafaela trained us and supervised us to be able to do this experiment.

To measure the bacterial colonization of the mosquitoes, we used pSB1C3-RFP transformed Pantoea agglomerans and E. coli and mixed them into the feeding sugar solution of the mosquitoes.


RFP transformed Pantoea agglomerans and E. coli on LBCl agar plates.


P. agglomerans-RFP and E. coli-RFP culture pellets.

An overnight bacterial culture was washed and resuspended in 5% sucrose, added to cotton pads and provided to adult mosquitoes for 3 days, after that, the cotton pads were replaced with new cotton pads containing a 5% sucrose solution.


Inoculation of bacterial suspension into the cotton pads. These were provided to adult mosquitoes on the top of the boxes.

For the detection of our chassis, we dissected the mosquitoes and homogenized the midguts and ovaries in PBS. From each sample we analysed 10 units. Then we looked for red fluorescence from the RFP protein using a fluorescence stereomicroscope. In addition to the midguts and ovaries we collected the saliva, which we also analised for fluorescence.


Collecting the mosquito's saliva. The mouthparts of the mosquitoes were pulled into pipet tipps with PBS and lef thee for 30 minutes.


Collecting the mosquito's saliva.


Dissecting the mosquitoes.


Visualization in fluorescence stereomicroscope.


Five ovaries of mosquitoes fed with sacarose to 24h (Negative control). A five ovaries microscope, B five ovaries with green filter, C five ovaries with red filter.


Six midgut of mosquitoes fed with sacarose to 24h (Negative control). A Six midgut microscope, B Six midgut with green filter, C Six midgut with red filter.


Six ovaries and two midgut of mosquitoes fed with P. agglomerans to 24h. A Six ovaries and two midgut microscope, B Six ovaries and two midgut with green filter, C Six ovaries and two midgut with red filter.


Seven midgut and two ovaries of mosquitoes fed with P. agglomerans to 24h. A Seven midgut and two ovaries microscope, B Seven midgut and two ovaries with green filter, C Seven midgut and two ovaries with red filter.


Nine ovaries of mosquitoes fed with E. coli to 24h. A Nine ovaries microscope, B Nine ovaries with green filter, C Nine ovaries with red filter.


Ten midgut of mosquitoes fed with E. coli to 24h. ATen midgut microscope, B Ten midgut with green filter, C Ten midgut with red filter, D two midgut with red filter from another spot.

We confirmed the colonization of P. agglomerans and E. coli strains in the midgut of female mosquitoes fed by 24h with the cotton pads containing the bacterials. The others samplings did not show the colonization of the mosquitoes.

References:

  1. Wang, Sibao, et al. "Fighting malaria with engineered symbiotic bacteria from vector mosquitoes." Proceedings of the National Academy of Sciences 109.31 (2012): 12734-12739. DOI: 10.1073/pnas.1204158109
  2. Wang, Sibao, et al. "Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria." Science 357.6358 (2017): 1399-1402. DOI: 10.1126/science.aan5478

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