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 plasmids (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 left. 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. The figures above contain bacteria that was streaked on media without the additional nutrients, and then was picked and restreaked again 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. The untransformed S.marcescens and Top 10 E. coli did not have the antibiotic resistance the plasmids coded for and therefore 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 could have taken longer time in 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. incubation.

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

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. There is minimum inhibitory concentration of antibiotics that was balanced by different concentrations in antibiotics. 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.

Transformation into Vibrio natriegens was only seen in the KAN plate, which again should have had 3 assemblies We see two types of colonies, and we know that the Vmax cells are the bigger ones and that the smaller size colonies are ones that took up the plasmid. This information was derived from instruction that came with a vibrio protocol . To see the protocol, click here. There were hints of color, but it was not that strong. This could be because of mutations that it takes longer for the color to appear. We believe that the lack of growth was due to the old promoter that we originally started out the kit with, GlpT. This promoter was not a broad host range promoter, therefore this might explain why the transformations were not as successful. We have begun to transition to a cp25 promoter that is broad range, and therefore will fit the purpose of the kit better. begun to transition We received the positive control from the Barrick lab and they were unsure if it was used, or if it had gone bad.

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.