Team:Humboldt Berlin/Contribution

Contributions

Improvement of minicell purification protocol and Calculator for centrifugation steps

Minicells need to be purified and separated from remaining parental cells to obtain quantifiable and reproducible samples for further downstreams analysis. We initially used the protocol provided by Jivrajani et al., 2013.¹

We found that the protocol by Jivrajani et al., 2013, is functioning and fairly optimized, however, we experienced a great loss of minicell yield after both or even a single filtration step (0.45 µm and 0.22 µm). Therefore, we altered the centrifugation steps to increase the minicell yield. Additionally, we set up a model to predict optimal centrifugation parameters such as time and speed.

By this, we found that using our optimized protocol, the filtration steps are not necessary anymore, which decreases the required time and price for a single purification. We think that this optimized protocol will help future iGEM teams interested in minicell related projects. It provides an easy to use, low effort and and cost effective approach to purify bacterial minicells.

Minicell purification (iGEM HU 2021 optimized, based on Jivrajani et al, 2013)

Bacterial Growth

1. Inoculate a single colony of a minicell-producing strain in 6 ml LB-medium (p.r.n. with supplements/antibiotic) → incubate ON at 37°C, 180 rpm.
2. Dilute ON culture in 500 ml LB-medium (supplement as needed) and grow at 37°C, 180 rpm until OD₆₀₀ of approximately 1 is reached.

Minicell Purification

• Centrifuge culture 2 times to separate parental cells from minicells
  • Speed and duration of centrifugation
    • (a) Use model to determine required centrifugation time according to culture volume and used beaker
    • (b) Or centrifuge 10 min @ 5,000 xg, RT
  • Transfer supernatant into fresh beaker, vortex
• Centrifuge 20 min @ 20,000 xg, RT → discard supernatant
• Resuspend pellet in 25 ml LB-medium → incubate 20 min @ 37°C, 180 rpm to reinitiate cell growth
• Add 200 µg/ml ampicillin and incubate 45 min @ 37°C, 180 rpm to lyse growing cells
• Centrifuge culture 2 times to remove as much cell debris as possible
  • Speed and duration of centrifugation
    • (a) Use model to determine required centrifugation time according to culture volume and used beaker
    • (b) Or centrifuge 10 min @ 5,000 xg, RT
  • Transfer supernatant into fresh tube, vortex
• Centrifuge 20 min @ 20,000 xg → discard supernatant
• Resuspend pellet in 2.5 ml LB-medium
• Filter sample through PVDF filter membrane with 0.45 µm pore size
  • Apply additional 0.5 ml LB-medium to filter to rinse the filter and push stuck minicells through the filter
• Check purity via microscopy and by plating 200 µl on LB-agar plate & incubate ON at 37°C

Fig. 1: Purification of minicells according to the original protocol and the improved protocol. Epi-fluorescence microscopy pictures are shown prior (left) and after purification (right).

Characterization of the PlldR (BBa_K1847008) from Escherichia coli in Salmonella Typhimurium.

The lactate inducible promoter PlldR (BBa_K1847008) from E. coli has been well characterized by different iGEM groups. As a part of our project, we intended to use this lactate inducible promoter to allow expression of a protein of interest in dependency of lactate as high lactate concentration (between 10-50 mM) are expected in the tumor microenvironment. However, this inducible promoter has not been characterized in our chassis organism, Salmonella Typhimurium. Therefore, we characterize the tunability of the promoter at different lactic acid concentrations. We hope this characterization might help future iGEM teams that use this promoter in Salmonella Typhimurium.

---

List of Sources

1. Jivrajani, M., Shrivastava, N. & Nivsarkar, M. A combination approach for rapid and high yielding purification of bacterial minicells. J. Microbiol. Methods 92, 340–343 (2013).