Contribution 2: How to conduct experiments with TetR-FROS system

How to make the donor cells:

To generate a suitable donor for the TetR-FROS system, first a constitutively expressed fluorescent protein gene must be integrated into the donor’s genome. This allows unambiguous donor identification during imaging. For example, we showed that it is possible to integrate the BFP gene into the CA434 genome using Tn7-mediated integration. However, any fluorescent protein can be inserted using any other available methods.

How to make the recipient cells:

Similarly, a constitutively expressed fluorescent protein gene must be inserted into the genome. However, this time it also needs to be fused to the TetR protein gene, so the resultant polypeptide is composed of both FP and TetR proteins. It should be ensured that different fluorescent proteins are used for the donor and recipient respectively to avoid any confusion during imaging.

How to make bacteria conjugate?

To induce conjugation, mix the donors and recipients and incubate them together for no less than 5 mins. The ratio of recipient to donor (R:D) can be manipulated to suit experimental aims. For example, we carried out conjugation assays with R:D ratios of 1:1, 3:1 and 10:1. Additionally, the medium used to mix donors and recipients can be either a microtube or the agarose pad itself. Both methods were shown to work and yield meaningful data in our experiments.

How to do imaging?

To image and gather data about conjugation, the first step is to create a suitable platform where your sample will be placed at. For our experiments, we made agarose pads on burnt cover slides and used a ONI Nanoimager to image the samples in the 405 nm and 488 nm channels, which are the blue and green channels respectively. The ONI Nanoimager also has the option for multi-acquisition which we used to image multiple fields of views. Alternatively, a microscope with a bigger field of view can be used to image a greater number of bacterial cells at a time. It is important to use the appropriate amount of power to image the fluorescent proteins, as to not bleach them and lose information.

How to do data analysis?

For data analysis, three sets of images for every field of view are needed. A bright-field image, a donor fluorescence image and a recipient fluorescence image. Use napari-bacseg to create segmentations of the bacterial cells which will require the bright-field image. Export the segmentation data in a .csv format and store it in the designated directory. Additionally, use napari-bacseg to create localisations for the foci in the recipient fluorescence image and store this data in a .csv file in the same directory as the segmentations. The parameters to identify localisations will have to be adjusted depending on the quality of the image, for example our parameters for localisation threshold was around 60,000.

One the relevant files have been generated, ensure that they all are present within the same directory including the donor fluorescence file. The next step is to use these files in Conj-assay to generate conjugation efficiency values. Conj-assay is quite particular with its file-naming conventions so use a basic naming system. To avoid these issues, we labelled all the files alphabetically to ensure the Conj-assay code did not break down while processing the files and also unchecked the ‘use custom regex’ setting in the Parameters tab. Additionally, replace the recipient filename in the name column of the localisation.csv with the donor filename. This filename should be consistent across the donor fluorescence image, localisation data and segmentation data.

Once all the files are correctly formatted, run Conj-assay and import Donor fluorescence image and localisation data. Process the data and then plot the Field-of-view and analyse what the program produces. Use the raw images and line up the plot to ensure that the program is correctly identifying donors, recipients and transconjugants. The cell fluorescence and localisation parameters will need to be adjusted in order to ensure data is interpreted/produced correctly. Save the results for further analysis.

See the example of processed data below:

Troubleshooting

The data analysis pipeline is sensitive to small typos and errors, and it can result in breaking of the code. Ensure that all above instructions are followed closely to ensure proper processing of data. Additionally, the alignment between Bright-Fields and the Fluorescent images has to be near perfect.

Any errors or misalignments between Bright-field images and recipient fluorescent images will result in the localisations being outside the segmentation, and the Conj-assay software will not pick up resulting in an unfaithful representation of the conjugation efficiency.

The experimental procedures are straightforward, with a few critical steps especially regarding making of M9 and washing of cells for imaging. Ensure a fresh batch of M9 is used day-to-day and that agar pads are flat and transparent, with no air bubbles or blemishes. It will reduce noise in the final images considerably, allowing for accurate localisation.

All appropriate proticols can be found on our Experiments page, including Mini-Prep, Gibson Assembly, Electroporation, Heat Shock, Plating & Culturing, and Imaging.

Software:

Napari-Bacseg - https://github.com/piedrro/napari-bacseg

Conj-Assay - https://github.com/alfbukys/conj_assay