Team:Georgia State/Results
iGEM GSU Experience with Team Lambert’s Homogenizer versus the Commercial Bead Beater
We did an Agrobacterium tumefaciens mediated transformation of pCB302-gfp-MBD plasmid into S. microadriaticum by first mildly disrupting the S. microadriaticum cells via bead beating and then creating a co-culture of the shaken algae cells and Agrobacterium tumefaciens carrying the plasmid of interest.
Following our collaboration with the Lambert students at the iGEM Presentation Bootcamp Meetup we hosted, we found that they had 3D-printed a low cost homogenizer of their own! So we decided to further collaborate by doing this transformation using a commercial bead beater and Lambert’s following this protocol.
Bead Beating Protocol and Observations Lambert vs. Commercial (link it T--Georgia State--protocolandobslambcom.pdf)
We found that Lambert’s homogenizer actually worked better than the commercial one, at least for our purposes in this experiment. When we used the commercial bead beater, some of the sample leaked out during the shaking process. It should be noted the commercial bead beater was defected to some degree and wasn’t fully operational. It shook back and forth and side to side when the motion should only be a back and forth one, but alas still worked. Following the bead beating process, we observed the S. microadriaticum cells underneath the microscope and found that there were no live or even visible cells after the commercial bead beating process. We hypothesize that it was too aggressive and caused the cells to explode. However, after using Lambert’s homogenizer for this bead beating protocol, we found that the S. microadriaticum was still alive and motile. Check out the live footage of the cells below!
We observed the cells once more about a week later and observed the same results.
In conclusion, for this experiment and for our purposes, Lambert’s bead beater proved to show greater cell viability following the bead beating process when compared to the commercial one we had in the lab, Cole-Parmer Mini bead beater, 115 VAC 60 Hz 5 in W x 7 in H x 10 in D (12.7 x 17.8 x 25.4 cm). Successful transformation of the S. microadriaticum is yet to be reported.
Dino III Plasmid and Codon Optimized RFP Ligation
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Linearized Dino III Plasmid
After obtaining high miniprep concentrations, our team ran a gel to determine if our DinoIII plasmid was successfully linearlized.
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pCB302-GFP in Agrobacterium tumefaciens
Our team planned on performing an Agrobacterium tumefaciens mediated transformation of pCB302-gfp-MBD plasmid into S. microadriaticum. However, to get to this step, we first needed to transform A. tumefaciens with the pCB302-GFP plasmid. After transformation, we performed a Colony PCR and ran a gel with the PCR products to confirm that the transformation was successful.
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After we confirmed the presence of pCB302-gfp, we sent it to Psomagen (previously Macrogen) for sequencing.
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Flow Cytometry
Our team tested out numerous transformation protocols for Symbiodinium microadriaticum. Because these cells are so sensitive and relatively easy to kill, we decided to perform a few “blank” transformations that contained no DNA to make sure our cells could survive the shock and recovery that comes with electroporation. One of the transformation protocols we tested was designed for Pichia (fungus).
Pichia Fungus Transformation Protocol (link pdf here) T--Georgia State--pichia.pdf
After allowing the cells to recover and grow for 3 days, we obtained flow cytometry data, with the help of one of our advisors.
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Our flow cytometry data is promising as we observed a few S. microadriaticum cells that are alive and displaying green fluorescence. This could mean that we successfully transformed Symbiodinium microadriaticum with the DinoIII-GFP plasmid.
Transformed Algae PCR
After we analyzed our flow cytometry data, we decided to run a PCR on the transformed algae to further confirm if the S. microadriaticum transformation was successful.
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Lonza Successful Pulse Codes
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Algae Growth Curves
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Future Plans
If our Lonza transformations for S. microadriaticum are successful, our team can take this project to the next step: coral culturing. With GSU’s campus being so close to The Georgia Aquarium, our team has the luxury of using coral specialist expertise to accurately and safely culture healthy corals in tanks. Kim Stone from The GA Aquarium has helped us design a way for these corals to uptake our modified algae by gradually increasing water temperatures in small increments until the coral have kicked out their original algal symbiont. This induced bleaching makes the coral weak and more likely to uptake our modified algae. Once the coral have been moved to a fresh tank, our modified symbiodinium microadriaticum carrying the DinoIII Codon Optimized RFP plasmid will be introduced and we gradually will bring the water down to an optimal temperature. If the coral uptake our algae successfully, we can increase the temperatures as we did the first time, to test if the algae are more resistant to being expelled by the corals.