dimeric FYVE

August 5-10

We started to conduct experiments of the Prevention stage. We acquired the Mus musculus embryonic cDNA from the Institute of Life Science in our school and successfully extracted the FYVE domain. The cloning cycles during these days were to insert the monomeric FYVE into standard backbone pSB1C3. The results were successful.  So for the next step, we tried to construct the dimeric FYVE.

8/11-8/15

In these days, we did lots of paper research about transfection of plant cells. We contacted an investigator of Academia Sinica and discussed about the experimental design of our project. We decided to use pSAT6, which was commonly used for transfection of plant cells, as our vector. We were also granted permission to conduct the experiment in the lab especially for plant transfection in Academia Sinica.

8/16-8/22

This week, we continued to construct the dimeric FYVE on pSB1C3. We constructed the dimeric FYVE with and without stop codon on the pSB1C3 plasmid. After constructing the dimeric FYVE without stop codon, we put the green fluorescence protein after it to create a fusion protein. We also managed to digest pSAT6, and successfully isolated the backbone for ligation. However, we failed to extract the dimeric form of FYVE from pSB1C3, thus we did many trials for troubleshooting and discovered flaws in our primer design.

8/23-8/29

We had lots of discussion this week and designed new primers to extract the dimeric FYVE from pSB1C3. To obtain a more accurate result, we run KOD PCR to proofread the sequence we want, and finally extracted the dimeric FYVE. We ligated dimeric FYVE and pSAT6 afterwards, in anticipation to complete our construct, which would later be transfected into tobacco BY-2 protoplasts.

8/30-9/5

It was confirmed that dimeric FYVE had successfully ligated with pSAT6. We also extracted the recombinant plasmids with the monomeric and dimeric form of FYVE for part submission and for making long-term stocks. We focused on contacting the research lab of Academia Sinica for transfection techniques and searching for the further experimental application of the PI3P inhibitor, Wortmannin.

9/6-9/12

We went to institution of Academia Sinica to do the transfection experiment. We failed to yield tobacco BY2 protoplast at first because the number of the plant cells used was not enough for transfection. After incubating for another day, we cultivated the BY-2 protoplast and transfected the construct into the cell. We also learned how to manipulate the fluorescence microscope. After the observation for a whole day, we got some great experimental results and wrapped up the prevention part!

Defensin

8/7-8/13

This week we started lab work in the curing section of our project. We do serial dilution to test the optimum concentration of the template we used, the IDT-synthesized Lm-def DNA sequence coding for defensin, to work with the primers (forward primer and reverse primer with stop codon) we designed while running PCR. After deciding the concentration level of the template, we did gel extraction after running KOD PCR for proofreading. We then inserted the part into pET29b, a vector designed for facilitating the purification of His-tagged protein, using E. coli BL21 (DE3) as chassis.

8/14-8/20

To prepare more stock for transformation, we made E. coli BL21 (DE3) competent cells in our lab. The function and activity of the competent cells were checked by transforming the pET29b empty vector and the recombinant plasmid with Lm-def (with stop codon) inserted into pET29b using kanamycin plates for antibiotics selection. The results were rather ideal. Apart from that, we worked on another set of primers (forward primer and reverse primer without stop codon) to extract and amplify the DNA fragment via KOD PCR. We eventually ligated the insert with the plasmid backbone pET29b after several trials. At the end of the week, we made long-term stocks of Lm-def (with stop codon)-pET29b and Lm-def (without stop codon)-pET29b.

8/21-8/27

This week, we prepared for the parts we would possibly need to transform Lm-def into pSB1C3 for part submission; namely, T7 with RBS (BBa_K731721) and T7 terminator (BBa_K525998). Besides, we also discussed on the next experiments that we would work on and designed the primers to add N-terminus 6x His-tag on to Lm-def (with stop codon)-pET29b and Lm-def (without stop codon)-pET29b, i. e. the recombinant plasmids that we had previously created.

8/28-9/3

The highlighted event this week is the growth rate assay to verify if the production of exogenous Lm-def affect the survival of our chassis. We prepare in advance LB broth and flasks; in the LB broth we added in IPTG for induction of protein expression and antibiotics, kanamycin, to be specific. Using Elisa reader, we tested the OD600 value of BL21 (DE3), BL21 (DE3) with pET29b empty vector, BL21 (DE3) with Lm-def (with stop codon) in pET29b, and BL21 (DE3) with Lm-def (without stop codon) in pET29b per 30 minutes to see their growth rate.

9/4-9/10

In this week, we started to add N-terminus 6x His-tag on to Lm-def (with stop codon)-pET29b and Lm-def (without stop codon)-pET29b upon acquisition of the primers we previously designed. We first acquired the template Lm-def (with stop codon and without stop codon) by running KOD PCR. Then we added cutting sites on them. Unfortunately, we always found an equal length band in our negative control, so we tried to change the ratio of dilution to our template and the concentration of Mg2+ in KOD PCR. And finally, it worked!

On the other hand, we also prepared liquid culture of T7 with RBS (BBa_K731721) and T7 terminator (BBa_K525998), so that we can start a new cloning cycle to insert Lm-def between T7 with RBS and T7 terminator on pSB1C3.

9/11-9/18

Happily, we back-inserted Lm-def (without stop codon) after T7 with RBS (BBa_K731721) this week. However, due to time limit, we failed to put T7 terminator behind them.

The major event this week was that we tested the function of defensin against P. infestans. To purify Lm-def, we lysed our cells and tested their concentrations at 600nm.

Next, to test the activity of defensin against P. infestans in vitro, we incubated in advance the oomycete acquired from Taiwan Agricultural Research Institute on rye agar plates at 18 degrees Celsius for ten days to acquire sporangia and hyphal pieces. We harvested P. infestans in sterile water, then 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. 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. 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. We then grow the plant under proper conditions for another week to observe changes in foliar tissues. Jane, who was in charge of contacting the research institutes helped with inoculation and microscopic inspection.