Demonstrate

Demonstrations via Conjugation

Conjugation into S.marcescens

When building the plasmid kit in E.coli, a model organism, electroporation was a convenient avenue for transforming plasmids into the host. However, electroporation protocols can vary for different bacteria species, and many may have no protocol developed for them at all. Rather than assuming the burden of developing one, researchers rely on conjugation. This is the transfer of a plasmid from one species of bacteria to the other, and happens in nature. We electroporated a GFP expressing assembly plasmid (BHR 902) from our kit into a strain of E.coli, called a Mu-free donor, that was a DAP auxotroph, so DAP had to be added to the growth media for the bacteria to survive. We then grew it along with S.marcescens at different ratios. Samples from the dual cultures were then grown on media without DAP, selecting for only S.marcescens. The S.marcescens that did grow on the media contained the plasmid, expressing the green fluorescence.

The results of the conjugation are shown in Figure 1. Although the green coloring of S.marcescens with the BHR kit plasmid (top) under UV light (right) is not as strong as some of the controls, the bacteria is clearly expressing the green phenotype, which it does not naturally have. Figure 1 contains bacteria that was streaked onto media without additional nutrients, which was then picked and re-streaked to ensure that no nutrients or colonies without plasmids remained. Because the first round of selective plating killed off the samples of only the MFD E.coli that needed the extra nutrients regardless of whether or not they contained a plasmid, they are not included on the plates above. Since the untransformed S.marcescens and Top 10 E. coli did not have the antibiotic resistance the plasmids coded for, they died on the media because it contained KAN antibiotic. The control plasmid contained a GFP and KAN resistance as well. However, it appears to have allowed the MFD strain to survive on the selective media, as traces can be seen in one of the control plasmid conjugation sections, as well as the sections where MFD plus the control plasmid was plated alone. However, because all the negative controls for the S.marcescens conjugation with a plasmid from the BHR kit died on the selective media, the results are not assumed to be invalidated. Ultimately, because the S.marcescens expressed the GFP from the BHR plasmid, we concluded that the Origin of Transfer (part type 7) was functional, allowing us to conjugate into other bacteria.

Demonstrating the One Tube Method

Electroporation into E. coli

At the core of the Broad Host Range Kit is the One Test Tube Method of testing plasmids in a host bacteria. The majority of people who get our kit will begin their experiments transforming a mixture of all the plasmids testing the origin of replication into a sample of their host bacteria. The idea is that through selective plating and use of visual reporters, researchers will quickly be able to determine which origins of replication wors in the non-model organisms. This 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 a fluorescent protein or chromoprotein specific and each color designates an origin. At a genomic level, a unique, non-coding sequence of DNA does the same thing, specifying which origin it is paired with. We created our one tube mixture of the initial assemblies by calculating an equimolar concentration.

The pictures to the right 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 a 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. This mixture is a preliminary version of what the one-tube reaction in our kit will contain, and tests the methodology of combining all plasmids, rather than segregating by antibiotic resistance. E.coli only grew with active reporter genes on the KAN and CAM plates but not CRB and TET. We believe we did not see growth for CRB and TET because the DNA concentration might have been too low or they may have needed to incubate either. The second mixture contained only 3 plasmids with the same antibiotic resistance, pAMB1+KAN, pMB1+KAN, and p15A+KAN, and was plated on a single KAN plate. This was done to focus on the effect of transforming multiple plasmids with the same antibiotic resistance, but different reporters and origins of replication. These tests allowed us to determine that the colored reporter system was an effective method of determining which plasmids were being expressed in the bacteria, but the absence of colonies on TET and CRB plates showed us that the higher concentrations of some plasmids relative to others is necessary so that the results are more apparent.

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 different antibiotic. The purpose here was so that only the E. coli that had picked up each specific assembly would grow on each plate. The results here were that only E. coli grew with active reporter genes on the KAN and CAM and this time on CRB as well. Their CRB plate was left to grow longer than ours, which might signify 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 and these were pAMB1+KAN, pMB1+KAN, and p15A+KAN, this was plated on a single KAN plate. This collaborative effort allowed us to see that results produced in other labs would be comparable to ours, and the Rice team was able to provide feedback on how we could improve the protocol we provided them.

Electroporation into Vibrio natriegens

Though we built the plasmid kit in E.coli because it is a reliable and easy to use model organism, that is not the bacteria it was designed for. To ensure that the plasmids and method of transforming many plasmids at once was applicable outside of E.coli we began testing the same preliminary version of the one tube mixture in other host organisms. The assemblies used in both E.coli electroporation and transformed it into Vibrio natriegens are pAMB1+AMP, pMB1+AMP, p15A+CAM, pAMB1+CAM, pAMB1+KAN, pMB1+KAN, P15a+KAN, p15A+TET. They were plated on media containing KAN, CRB, or CAM. There was a second transformation that involved a positive control provided by the Vmax kit. We received this positive control from the Barrick lab and they were unsure if it was used, or if it had gone bad.note:what is this actually telling the reader? maybe just delete and say quality of controls was suspect There is minimum inhibitory concentration of antibiotics that was balanced by different concentrations in antibiotics. note:is this necessary to mention Colony growth was seen only in KAN and CAM plates, however, there was no clear presence of the reporter colors on either plate. Additionally, the KAN plate has two size colonies growing.

Transformation into Vibrio natriegens was only evident in the KAN plate, to which three assembly plasmids contain the antibiotic resistance gene. The protocol for transforming into Vmax cells (electrocompetent Vibrio 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. To see the protocol, click here. There were hints of color in the smaller colonies, but what was observed could not be considered conclusive evidence of reporter genes being expressed. This could be because of mutations that it takes longer for the color to appear. note:what? 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 Vibrio 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 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.

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 left.

Demonstration of Chromoproteins

A plate of all the chromoproteins available in our lab was made to show what expected results from a successful transformation using the BHR Kit. Seven chromoproteins, BCP, PCP, pink, GFP, GCP, E2C, and RCP, were grown in overnight cultures from a glycerol stock. 100µl of each of the seven O/N cultures were mixed together and diluted to a 1:100,000. The mix had to be diluted because successful transformations usually have few colonies present. Having a few individual colonies also makes it easier to distinguish between the different chromoproteins. 50µl of this chromoprotein mix was then streaked out on an LB only plate since the pink chromoprotein has KAN resistance while the rest have CAM resistance. The plate was left to grow overnight in a 37°C incubator however, some chromoproteins take another day or two to be strongly expressed by the colonies. The plate to the right shows the plate on a black background and under a blue UV light. Some chromoproteins, like E2C and GFP, are more easily identified under UV light because they fluoresce or change colors. The figure to the left also shows the plate on a white background making it easier to identify the E2C colonies. RCP was also present on the plate but was not easily identifiable in the photos taken.