Team:Austin UTexas/Results/Electroporations

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Electroporations

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Part Plasmids Assembly Plasmids Transformations Conjugations

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Results of One-Tube Electroporation

At the core of the Broad Host Range Kit is the One Test Tube Method of testing plasmids in a host bacteria. This is how the majority of people who receive our kit will begin their experiments. Using our kit, a researcher will be able to transform multiple assembled plasmids at once, and then through selective plating determine which plasmids work in their non-model organisms. Specifically, this will allow the researcher to identify what origin of replication works, which is the first step in building a plasmid to genetically engineering an organism. Results will be confirmed by one of two ways: assessing the phenotype produced by the reporter gene or sequencing at the barcode region. The reporter is either fluorescent protein-specific or chromoprotein-specific, and each color designates an origin. At the DNA level, a unique, non-coding Barcode sequence of DNA does the same thing, specifying which origin is on the plasmid. Our one tube mixture of initial assemblies contains an equimolar concentration of plasmids.

Electroporation into E. coli

Figure 1: Transformation of the One-Tube Reactions into E. coli Via Electroporation. Above are plates containing the transformants from a one-tube reaction. Within this reaction, many different assembly plasmids are added to a competent cell mixture. The various reporter genes will help to distinguish which assemblies were successfully transformed. This can be visualized in the far-right plate where both crimson and green colonies can be seen.

The plates in Figure 1 represent our first trials to see if the one tube method would work. The first mixture had 8 different assemblies (pAMB1+AMP, pMB1+AMP, p15A+CAM, pAMB1+CAM, pAMB1+KAN, pMB1+KAN, P15a+KAN, p15A+TET) and bacteria transformed with it was plated on 4 different plates, each with different antibiotic to select for E. coli expressing a plasmid with the corresponding antibiotic resistance. The p15a and pMB1 have a green fluorescent phenotype and the pAMB1 is paired with E2-crimson reporter, which appears black in normal light and red under UV light. The results show that E.coli only grew with active reporter genes on the KAN and CAM plates, but did not grow on the CRB and TET plates. The second mixture contained only 3 plasmids, pAMB1+KAN, pMB1+KAN, and p15A+KAN, and was plated on a single KAN plate. We believe we did not see growth for CRB and TET because the DNA concentration might have been too low or the incubation time was too short.

Figure 2: Collaboration with Rice University — One-Tube Reactions. The plates above represent Rice’s attempts to utilize the one-tube reactions. The plates exhibit multiple morphologies, demonstrating that multiple assemblies have been successfully introduced into cells. As in Figure 1, E. coli was plated onto LB plates with varying antibiotics.

Rice University performed the same initial tests independently. One tube had 8 different assemblies (pAMB1+AMP, pMB1+AMP, p15A+CAM, pAMB1+CAM, pAMB1+KAN, pMB1+KAN, P15a+KAN, p15A+TET). This reaction was plated on 4 different plates, each with a different antibiotic. This way, only the E. coli that had picked up each specific assembly would grow on the respective plate. Figure 2 shows that E. coli only grew with active reporter genes on the KAN and CAM, as well as CRB. Their CRB plate was left to grow longer than ours, which might be why they saw positive results. TET was not plated because Rice University did not have the antibiotic available. The second tube contained only 3 reactions, which were pAMB1+KAN, pMB1+KAN, and p15A+KAN; this was plated on a single KAN plate.

Electroporation into Vibrio natriegens

We used the same assemblies in reaction tube 1 from E.coli and transformed into Vibrio natriegens. The reaction contained 8 assemblies. These assemblies were pAMB1+AMP, pMB1+AMP, p15A+CAM, pAMB1+CAM, pAMB1+KAN, pMB1+KAN, P15a+KAN, p15A+TET. They were plated only in KAN, CRB, and CAM. There was a second transformation that involved a positive control provided by the Vmax kit. Colony growth was seen only in KAN and CAM plates. No type of reporter color was seen in any plate. The KAN plate has two size colonies growing.

Figure 3: Transformation of One-Tube Reaction Into Vibrio natriegens. Using a single-tube plasmid mix, non-model organism, Vibrio natriegens was transformed. Growth can be seen on this kanamycin-containing LB plate. It should be noted that two distinct colony morphologies are present. Neither showed distinct colored or fluorescent phenotypes — some colonies were lightly colored.

Transformation into Vibrio natriegens was only really seen in the KAN plate (Figure 3), to which three assembly plasmids contain the antibiotic resistance gene. The protocol for transforming into Vmax cells (electrocompetent V. natriegens) sites the presence of two types of colonies as evidence for successful transformation, the smaller size colonies being the ones with the extra burden of expressing the additional plasmid. This would lead us to believe that the transformation was succesful. We received the positive control from the Barrick lab who told us that the quality of controls was not optimal, which might explain its lack of functionality. There were hints of color in the smaller colonies that grew in KAN, but what was observed could not be considered conclusive evidence of reporter genes being expressed. This lack of color in the KAN plate, and lack of growth in other plates could be due to the inefficiency of the promoter that is currently present in the completed assemblies. Up until this point, we had been making our assemblies with the GlpT promoter. However, this promoter does not have a particularly broad host range, which may explain why the transformations did not appear to be as successful. The lack of reporter expression in V. natriegens has led us to begin transitioning to using the CP25 promoter in our assemblies and bridge parts, because it is considerably more broad host range and has a higher expression rate, and therefore will be more compatible kit design and use. .

We have several options for next steps. These involve picking colonies and plating them on new plates to see if they grow normally, and/or sequencing colonies for their barcodes which are specific to origins. This will allow us to check which origins are working. From there on we might want to retransform for further verification.

Electroporation into Mu-Free Donor E. coli

In order to test the BHR kit’s function in a diverse array of organisms, we demonstrated how a one tube plasmid mix, which contained multiple assembly plasmids, could be transformed into DAP auxotrophic Mu-Free Donor (MFD) E. coli cells. These transformed Mu-Free Donors would ultimately be used to conjugate with non-model organisms in which electroporation is not a viable option for transformation. When attempting to conjugate with a target non-model organism, donor cells carrying the BHR plasmids must first be created. Rather than performing an independent transformation with the donor cells for each assembly plasmid, it is more time-efficient to perform a single procedure that transforms all of the assembly plasmids into the donor cells that is followed by a selection of the recovered cells. In this demonstration, we show the how the one tube transformation can easily be applied to Mu-Free Donors for conjugation.

Figure 4: Transformation of One-Tube Reaction Into Mu-Free Donor E. coli. The above photos show LB plates with varying types of antibiotics. MFD E. coli were electroporated, recovered in SOC media, and plated. Although very few different reporters can be seen (sGFP dominates each plate except on CRB), it is possible that multiple plasmids were transformed into cells on the plates.

Electrocompetent MFU donors were electroporated with a single mix of DNA containing eight assembly plasmids. This eight-assembly mix consisted of two plasmids conferring AMP resistance, two plasmids conferring CAM resistance, three plasmids conferring KAN resistance, and a single plasmid conferring TET resistance. Therefore, the selective plates used to plate the transformation recoveries were AMP, CAM, KAN, and TET plates. The antibiotics were added to the media at a 1:1000 ratio, and the media was supplemented with DAP at 6 µL DAP per ml of media. The electrocompetent MFD cells were transformed via electroporation on settings suitable for E. coli and allowed to recover for an hour at 37°C. 100 µL of the recovery was plated on each selective plate and incubated overnight. The results of this demonstration are shown in Figure 4.

Future Directions

Once we have created enough unique origins of replications, we can use the arsenal of reporters we have in our kit, which include Blue Chromoprotein (BCP), Pink Chromoprotein (PiCP), Purple Chromoprotein (PCP), Green Fluorescent Protein (GFP), E2 Crimson (E2C), Red Chromoprotein (RCP), and Green Chromoprotein (GCP). In theory, we could test 7 different origins of replication in parallel in our organism of interest and be able to visually screen for which ORI(s) work with the host's cellular machinery.

Figure 5: Part plasmids containing the various reporter genes in our kit. Left to right: BCP, PiCP, PCP, GFP, E2C, RCP, and GCP.