iGEM 2021 — Team Humboldt_Berlin

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# Team:Humboldt Berlin/Notebook

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## Notebook

## Lab Notebook

| Date | Description |
| --- | --- |
| 15.04.2020 - 30/08/2021 | Drylabfirst introduction to use glycoproteins as surface marker/specificity marker, research to possible immune responses ⇒ lectins as final surface proteins |
| 05/08/2020 - 26/03/2021 | Drylab initial research to minicells (how to create them using the MinCDE system, advantages to normal *Salmonella* cells, virulence, purification) |
| 16/11/2020 - 03/03/2021 | Wetlabconstruction of ST14028 MvP103 *sseC::aphT* Para*::hilA+* Δ*minD*::FRT strain |
| 09/12/2020 | succesfull insertion of Δ*minD::fcf* in EM11446   ![succesfullinsertionofDminD::FCFinEM11446](https://static.igem.org/mediawiki/2021/5/50/T--Humboldt_Berlin--Target_TaxiDminDFCFCEM11446.jpeg) |
| 02/02/2021 - 29/05/21 | Drylab research for an alternative way to create mini cells using the *ftsZ* gene (discontinued) |
| 01/03/2021 - 09/10/2021 | Wetlabconstruction of a plasmid for PlldR (BBa\_K1847008) characterization in *Salmonella* Typhimurium |
| 30/09/2021 | successful cloning of pSB1C3-PlldR-*gfp* in *Salmonella* (EM7653)   ![successful cloning of pSB1C3-PlldR-GFP in EM7653 ](https://static.igem.org/mediawiki/2021/3/34/T--Humboldt_Berlin--Target_Taxi_pSB1C3-PlldR-GFP_in_EM7653.png) |
| 07/06/2021 | Wetlab construction of *mCherry* and *gfp* expressing ΔminD::FRT strain for visualization of minicells |
| 10/06/2021 - 24/06/2021 | Wetlab visualization of minicells |
| 11/06/2020 | first mini cell purification results using *Salmonella* (EM7653), EM12053)   ![1st mini cell purification results using EM12052 and EM12053 ](https://static.igem.org/mediawiki/2021/c/ce/T--Humboldt_Berlin--Target_Taxi_first_purification.png) |
| 15/06/2021- 15/08/2021 | Drylab Design of pSB1C3-derived pKF01, the empty backbone that expresses *thyA* and *lldR* constitutively from the synthetic promoter Psyn-thyA. Thereupon, the plasmid pKF02 was designed in silico. pKF02 is supposed to express *sicP-sptP-*pep8** from the lactate-inducible promoter PlldR. |
| 11/07/2021 | Drylab introduction to the protocol written by Joyce Karlinsey and optimized for *Salmonella* Typhimurium LT2 in the lab of Dr. Kelly T. for allelic replacement using lambda-RED and *tetRA* replacement |
| 20/07/2021 | Drylab introduction to using agarpads for fluorescence microscopy |
| 09/08/2021 - 26/09/2021 | Wetlab visualization of Minicells II - DiSC and FM4-64 |
| 09/08/2021 | second minicell purification results using *Salmonella* (EM7653)   ![Purification results using an optimized mini cell purification pre- and post-filtration in EM12052.](https://static.igem.org/mediawiki/2021/d/d9/T--Humboldt_Berlin--Target_Taxi_second_purification.png) |
| 22/09/2021 | purification results using an optimized mini cell purification pre- and post-filtration in *Salmonella* (EM7653)   ![Purification results using an optimized mini cell purification pre- and post-filtration in EM12052.](https://static.igem.org/mediawiki/2021/7/76/T--Humboldt_Berlin--Target_Taxi_optimization_purification.png) |
| 12/08/2021 | Drylab introduction to *galE* deletion via *tetRA* insertion and replacement |
| 12/08/2021 | wetlab *galE* deletion via *tetRA* insertion and replacement |
| 14/08/2021 - 09/10/2021 | Wetlab cloning the *pep8*-Biobrick and PlldR*-sicP-sptP*-Biobrick into the pSB1A3 backbone |
| 20/09/2021 | succesfull insertion of PlldR*-sicP-sptP* into pSB1A3   ![ succesfull insertion of PlldR-sicP-sptP into pSB1A3](https://static.igem.org/mediawiki/2021/2/2b/T--Humboldt_Berlin--Target_Taxi_plldr_sicP_sptP.png) |
| 14/08/2021 - 14/09/2021 | Wetlab construction of ST14028 MvP103 *sseC::aphT DthyA* for pKF01 cloning |
| 26/08/2021 | successful replacement of *ThyA*-Gene with *tetRA* cassette   ![ successful replacement of ThyA-Gene with TetRA cassette](https://static.igem.org/mediawiki/2021/e/e4/T--Humboldt_Berlin--Target_Taxi_thyA_tetRA.png) |
| 14/08/2021 - 07/09/2021 | Wetlab construction of *DgalE* mutants in ParaBAD::*hilA* strains |
| 23/08/2021 | successful insertion of the *tetRA* cassette within *galE*   ![  insertion of tetRA within galE](https://static.igem.org/mediawiki/2021/3/37/T--Humboldt_Berlin--Target_Taxi_galE_tetRA.png) |
| 07/09/2020 | D*galE*-clean was introduced at the right position 07/09/2021   ![ Colony PCR - check if DgalE-clean was introduced at the right position](https://static.igem.org/mediawiki/2021/2/21/T--Humboldt_Berlin--Target_Taxi_dgalEclean.png) |
| 22/08/2021 - 26/09/2021 | Wetlab visualization of Minicells II - DiSC and FM4-64 |
| 23/08/2021 | successful insertion of the TetRA cassette within *galE* |
| 25/08/2021 | Drylab getting familiar with the DNA assembly - BioBrick NEB |
| 24/09/21 - 26/09/21 | Drylab design of the Codon optimized E\_0840 part for *Salmonella* Typhimurium LT2 |
| 25/09/2021 - 26/09/2021 | Wetlabminicell purification to determine minicell age distribution |
| 26/09/2021 | determine the minicell age distribution by quantifiying mini cell number based on OD values at given time points using *Salmonella* (EM7653)   ![ Minicell purification to determine Minicell age distribution](https://static.igem.org/mediawiki/2021/f/fd/T--Humboldt_Berlin--Target_Taxi_age_distribution.png) |
| 25/09/2021 - 05/10/2021 | Wetlabconstruction of plasmid for PlldR (BBa\_K1847008) characterization in *Salmonella* Typhimurium |
| 04/10/2021 | successfull construction of pSB1C3-PlldR-*gfp* |
| 27/09/2021 | Wetlabgrowth curve of minicell producing strains |
| 30/09/2021 - 09/10/2021 | Wetlablife span/membrane integrity of minicells via Disc assay   ![  Life span/Membrane integrity of minicells via Disc assay ](https://static.igem.org/mediawiki/2021/a/a1/T--Humboldt_Berlin--Target_Taxi_DiSC.png) |
| 01/10/21 | Drylab design of PlldR-*gfp* (E\_0840) part to test the inducible promoter |
| 01/10/2021 - 16/10/2021 | Wetlabassessment of production rate and size determination of minicells |
| 11/10/2021 | production timelapse of minicells in our Δ*minD* *Salmonella* Typhimurium strain   ![ production timelapse of minicells in our ΔminD Salmonella typhimurium strain](https://static.igem.org/mediawiki/2021/b/be/T--Humboldt_Berlin--Target_Taxi_production_timelaps.gif) |
| 09/10/2021 | Wetlabcloning *AIDA-rBC2LCN* in pSB1C3 |
| 12/10/2021 - 21/10/2021 | Wetlabcharacterization of the PlldR-*gfp* promoter in *Salmonella* Typhimurium |
| 14/10/2021 | pKF01-PlldR-*gfp* cloning succeeded |

## Modelling Notebook

| Date | Description |
| --- | --- |
| April 2020 | collecting modeling and implementation ideas |
| May 2021 | modeling the metabolism rate as well as the lifespan of minicells, design of experiments to determine the lifespan (for example Proton Motive Force) |
| June 2021 | outlines of population models for minicells by differential equations |
| July 2021 | statistical modeling, assumption of log-normal distribution for the size of the minicells, binomial distribution for number of generated minicells |
| August/September 2021 | Data analysis with CellProfiler, Fiji and Python |
| October 2021 | Analysis of sedimentation rate from laboratory is coupled to modeling, improvement of population model |

## Public Relations Notebook

| Date | Description |
| --- | --- |
| Mar 2020 | Working on the TargetTaxi Logo |
| Apr 2020 | Announcement of "TargetTaxi" on Instagram and Facebook with the first Zoom-Team Photo |
| Oct 2020 | Foundation of the PR Team: Philipp Kittler, Olyssa Sperling |
| Nov 2020 | Working out a plan to explain our Lab Work |
| Dec 2020 | Salmonella-as-SantaChristmas Post on Instagram |
| Jan 2021 | Working on a TargetTaxi Flyer |
| Apr 2021 | "The Minicell in a Nutshell" |
| May 2021 | "Phase one: production ofminicells", "Phase two:secretion of proteins and adhesion to cancer cells", "Phase three: apoptosis of the cancer cell", “We are participating!” Announcement on Instagram |
| Jun 2021 | New members in the PR Team: Vincent Fölsche and Martje Grensemann, Zoom-Meeting with iGEM Iiserb Bhopal from India about a Collaboration, Announcement that we are finally allowed to work in the lab, Zoom-Meeting with UBC iGEM from Vancouver, Canada, about our Collaborationon *Salmonella* Typhimurium |
| Jul 2021 | Planning and shooting scenes for our Promotion Video |
| Aug 2021 | The publishing of our Promotion Video, Meeting with Promega about Sponsorship Ideas |
| Sep 2021 | Introduction of the PR Sub Team on Instagram and Facebook, Planning scenes and structure of our Presentation Video |
| Oct 2021 | Collaboration Posts with UBC iGEM on Cancer and Salmonella, Shooting Scenes for our Presentation Video, Introduction of theLab and Modelling Sub Teams on Instagram and Facebook, Publishing of our Presentation Video |

## Fundraising Notebook

| Date | Description |
| --- | --- |
| Jan 2020 | brainstorming who we could contact (biotech and pharmaceutical companies, foundations, crowdfunding maybe) and researching contact information |
| Feb 2020 | making a list of things we should address during phone calls and making a template for emails to potential sponsors |
| Mar 2020 | making first phone calls and sending emails |
| Oct 2020 | deciding on different levels of rewards for sponsors based on how much money they give |
| Jan 2021 | designing a flyer for sponsors, requiring Eppendorf’s and NEB's support |
| Feb 2021 | requiring Roth’s support |
| Mar 2021 | requiring IBA lifesciences' support |
| Mar 2021 | requiring Promega's support |
| Sep 2021 | requiring Microsynth Seqlab’s support |
| Oct 2021 | requiring Dunn Labortechnik's, BioRender's and Gilson's support |

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# Team:Humboldt Berlin/Engineering

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## Engineering Success

### Inducible Promoter System

In most synthetic biology projects, controllable and tunable transcription is crucial for the success. Often genetic circuits rely on a sharp and time resolved hierarchy of gene activation. For this, inducible promoters are an inventibly option to achieve the upper mentioned prerequisites. In our project, we also encountered the need to apply an inducible promoter for our expression system (link out to Pep8 expression). For this we first tried a construct containing an Arabinose inducible promoter ParaBAD fused to GFPmut2 (ParaBAD::GFPmut2) in the pGAP18 backbone. The araBAD promoter allows the L-arabinose induced expression of the fused construct.[1](#Guzman1) This way, gene expression can be controlled and adapted to the experimental requirements based on the applied L-arabinose concentration and glucose depletion.[1](#Guzman1) However, after successful cloning and transformation of the construct, no GFPmut2 expression was observed upon L-arabinose addition.

After extensive literature search, we concluded that potential uptake of Arabinose into minicells could be problematic due to the lack of importers. L-arabinose cannot penetrate the bacterial inner membrane.

Thus, L-arabinose intracellular uptake is transporter dependent by either a low affinity transporter araE (protein symporter) or high an affinity transporter araFGH (ABC-transporter).[2](#Luo1) Furthermore, in contrast to the constitutively expressed and incessantly present low affinity transporter, the high affinity transporter is only formed upon L-arabinose sensing.[2](#Luo1) However, minicells do not harbor a chromosome on which araFGH is encoded.

Also, and equally important, minicells possess limited transcriptional and translational capabilities due to their small size.[3](#Farley1) We therefore consider the possibility that minicells are unable to effectively transcribe, translate and assemble the high affinity transporters in sufficient quantity. Finally, ATP is also limited in minicells which could further influence the effectiveness of ATP driven transporters in general.

Due to the presented reasons, we switched to a tetracycline-controlled transcriptional activation system (pTet-tetR) fused to our *Salmonella* codon harmonized GFP ([BBa\_K3861027](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3861027); [BBa\_K3861011 + BBa\_K3861019]). The tetR system is well characterized and may be the most used gene expression controlling promoter system to date.[4](#Gossen1) There are two variants of this promoter-induction system, called Tet-On and Tet-Off.[5](#Baron1),[6](#Berens1) We use the Tet-On variant that selectively activates expression in the presence of tetracyclines. Important for our needs is the fact that tetracycline (or Anhydro-tetracycline) can passively penetrate bacterial membranes and no active transportes systems is crucial for this inducible expression system.

---

**List of Sources**

1. Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).
2. Luo, Y., Zhang, T. & Wu, H. The transport and mediation mechanisms of the common sugars in Escherichia coli. Biotechnol. Adv. 32, 905–919 (2014).
3. Farley, M. M., Hu, B., Margolin, W. & Liu, J. Minicells, Back in Fashion. J. Bacteriol. 198, 1186–1195 (2016).
4. Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. U. S. A. 89, 5547–5551 (1992).
5. Baron, U. & Bujard, H. Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods Enzymol. 327, 401–421 (2000).
6. Berens, C. & Hillen, W. Gene regulation by tetracyclines. Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes. Eur. J. Biochem. 270, 3109–3121 (2003).

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## Experiments

### DNA assembly of [RCF10 / RCF25] BioBricks based on NEB protocols

Protocol to assemble a composite part designated into a vector backbone.

#### A. Digestion

1. Digest upstream BioBrick with EcoRI-HF® and SpeI. For translational fusions, EcoRI-HF and AgeI were used.

   |  |  |
   | --- | --- |
   | Upstream Part | 1 µg |
   | EcoRI-HF | 1 µl |
   | Spel or AgeI | 1 µl |
   | 10X NEBuffer 2.1 | 5 µl |
   | H2O | ad 50 µl |
2. Digest downstream BioBrick with XbaI and PstI. For translational fusions, NgoMIV and PstI were used.

   |  |  |
   | --- | --- |
   | Upstream Part Plasmid | 1 µg |
   | Xbal or NgoMIV | 1 µl |
   | Pstl | 1 µl |
   | 10X NEBuffer 2.1 | 5 µl |
   | H2O | ad 50 µl |
3. Digest the vector EcoRI-HF® and PstI.

   |  |  |
   | --- | --- |
   | Destination Plasmid DNA | 1-4 µg |
   | EcoRI-HF | 1 µl |
   | Pstl | 1 µl |
   | 10X NEBuffer 2.1 | 5 µl |
   | H2O | ad 50 µl |
4. Incubate all three restriction digest reactions at 37°C for 1 h. 1 µl of Quick CIP was added to the backbone restriction reaction for dephosphorylation.
5. Digested products were checked by gel electrophoresis and subsequently purified from the gel. Inserts were purified by using the PCR clean-up Kit (Macherey-Nagel / Promega).

#### B. Ligation

For ligation of two BioBricks into a designated backbone, the following procedure was performed:

|  |  |
| --- | --- |
| Part 1 digestion | 1:3 (Vector:insert ratio) |
| Part 2 digestion | 1:3 (Vector:insert ratio) |
| Destination Plasmid digestion | 100 ng |
| 10X T4 DNA Ligase Buffer | 2 µl |
| T4 DNA Ligase | 1 µl |
| H2O | ad 20 µl |

Incubate at room temperature for 30 min and then heat inactivate at 80°C for 20 min. Purify using PCR clean-up kit and transform into an appropriate recipient.

### Transformation via electroporation

#### 1. Production and preparation of recipient (e.g. Dh10alpha *E.* coli)

Main culture was started using a 1:100 overnight culture:main culture (v/v) and incubated at 37°C until OD600 of ∼ 0.6 was reached. Cells were harvested via centrifugation for 5 min at 10.000 xg and washed two times in 25 mL ice-cold MQ-H2O. After washing, cells were resuspended in 200 µl MQ-H2O and placed on ice.

#### 2. Electroporation and confirmation

5 µl linear DNA or 1 µl plasmid DNA was mixed with 50 µl competent cells and transferred into ice-cold electroporation cuvettes. Samples were electroporated and immediately transferred into 1 ml LB and incubated for 1 h at 37°C. Mixture was spread onto appropriate (dependent on backbone applied) antibiotic LB plates followed by overnight incubation at 37°C. The next day, plates were restreaked to gain single colonies.

### Allelic replacement using lambda-RED and tetRA replacement

This protocol, written by Joyce Karlinsey, has been optimized for *Salmonella* Typhimurium LT2 in the lab of Dr. Kelly T. Hughes. Cite: *Genetic Transplantation: Salmonella enterica Serovar Typhimurium as a Host To Study Sigma Factor and Anti-Sigma Factor Interactions in Genetically Intractable Systems, Karlinsey, Hughes.*

### Measuring expression capacity of minicells

For the characterization of the expression capacity of minicells, the fluorescence of mCherry was measured over time. Minicells were purified from a strain that constitutively expressed mCherry and from a control strain which did not express mCherry. 100 µl of the purified minicells were distributed into a 96 well plate. As a control, translation of mCherry was inhibited by the addition of tetracycline to a final concentration of 30µg/ml. For each control, as well as for the mCherry expressing minicells, the analysis was performed in three different technical replicates. Fluorescence was measured every 5 minutes for 6h at 37°C under constant shaking. mCherry excitation was performed at 590 nm excitation, 9 nm bandwidth; emission was measured at 620 nm, 20 nm bandwidth.

### PCR

#### Amplification of Inserts for Cloning and Cassettes for Homologous Recombination

A Q5 high fidelity polymerase from New England Biolabs was used for this purpose, as it has a very low error rate even with large amplifications. The procedure is shown in the table below.

|  |  |
| --- | --- |
| Component | 50 µl reaction |
| Q5 High-Fidelity 2x Master Mix | 25 µl |
| 10 µM forward primer | 2.5 µl |
| 10 µM reverse primer | 2.5 µl |
| Template DNA | variable |
| Nuclease-free H2O | ad 50 µl |

|  |  |  |
| --- | --- | --- |
| Initial Denaturation | 98°C | 30s |
| 35 cycles | 98°C | 10s |
| NEB Tm Calculator | 30s |
| 72°C | 30s/kb |
| Final Extension | 72°C | 2 min |
| Hold | 4°C | ∞ |

#### Colony PCR

For colony PCR, lab internal manufactured Taq polymerase was used. Instead of the isolated plasmid to be amplified, some cell material of a transformant colony was used as a template, from which the plasmid to be amplified was released by prolonged initial denaturation.

|  | Master Mix [µl] | |
| --- | --- | --- |
|  | Stock | 1x |
| 1x PCR buffer | 5x | 4.0 |
| 1.5 mM MgCl2 | 25 mM | 1.2 |
| 800 µM dNTP’s | 20 mM | 0.8 |
| 0.5 µM primer 1 | 10 µM | 1.0 |
| 0.5 µM primer 2 | 10 µM | 1.0 |
| Go-Taq Polymerase (U) | 5 U/µl | 0.2 |
| Template DNA |  | Picked colony |
| Nuclease-free H2O |  | ad to 20 µl |

| Cycle | Time | Temperature |  |
| --- | --- | --- | --- |
| initial denaturation | 3 min | 95 °C |  |
| denaturation | 5-15 sec | 95 °C |  |
| annealing | 30 sec | Ann | 35 cycles |
| extension | 60 sec/1kb | 72 °C |  |
| final extension | 5 min | 72 °C |  |
| soak | ∞ | 12 °C |  |

#### Agarose Gel Electrophoresis

To separate DNA based on size, gel electrophoresis was applied to agarose at a concentration of 1% (depending on desired fragment size) [w/v]. The 100 bp extended ladder from ROTH was used to determine fragment size. Separation was performed at 100 V for 25 - 45 min, and the gel was placed in a 0.1% [w/v] GelRed solution bath for DNA staining. GelRed intercalates in DNA, which makes DNA visible under UV light.

#### Isolation of Plasmid DNA

A total of 4 ml of the overnight culture was used to obtain plasmid DNA using the “Monarch® Plasmid Miniprep Kit“ from NEB.

#### Microscopy - time-lapse

An overnight culture of a minicell producing strain, which expressess mCherry from a constitutive promoter was diluted 1:100 in LB medium and grown at 37 °C until the late log phase was reached. The culture was diluted to an OD600 of 0.1 and 2.5 µl were spread on an agarose pad in a Gene Frame (Thermofisher scientific). Cells were visualized every 15 min for a period of 5 h. To image mCherry a 640 nm laser set to 5% was used. Samples were exposed to the laser for 100 ms.

#### Microscopy - PMF Measurement

Minicells expressing GFP under regulation of the lldR promoter were purified according to the protocol “purification of minicells”. Purified minicells were sedimented at 20,000 xg for 1 min and resuspended in 600 µl LB medium. The resuspended minicells were incubated at 37 °C, 700 rpm. At each given time point indicated: 50 µl of the minicell sample were taken and mixed with 450 µl 1x PBS, supplemented with 0.2% glucose.

3,3'-Dipropylthiadicarbocyanine Iodide (DiSC3(5)) was added to the culture to a final concentration of 0.2 mM and incubated for 5 min at 37 °C under constant shaking. A sample of the culture was then spread on an agarose pad in a gene frame (Thermo Fisher scientific) and imaged. To image GFP and DiSC3(5), a 488 nm and a 640 nm laser set to 5% intensity were used. GFP was imaged using an exposure time of 100 ms and DiSC3(5) of 200 ms.

#### Microscopy - Cell Membrane Staining and High Resolution Microscopy

FM 4-64 was added to a final concentration of 5µg/mL to a sample of either purified minicells or a growing culture of minicell-producing strain. The sample was then incubated for 30 min at 37 °C and 700 rpm. Subsequently, the minicells were centrifuged at 20,000 xg, for 1 min and the supernatant was discarded. The pellet was resuspended in 1x PBS of the initial volume. Cells were then imaged using the STED microscope from Abberior.

#### Characterization of Promoter Activity

For characterization of promoter activity, bacteria expressing GFP from the lldR promoter were grown to early log phase and subsequently diluted to an OD600 of 0.01. 100 µl aliquots of the cell culture were then distributed in a flat bottom black plate (Greiner) in triplicates for each lactic acid concentration. Plate was then incubated for 1 h at 37 °C under constant shaking. After 1 h incubation, expression of GFP was induced by addition of lactic acid to different concentrations ( 0 mM, 0.2 mM, 0.4 mM, 0.8 mM, 1.6 mM, 3.2 mM, 6.4 mM, 12.5 mM, 25 mM and 50 mM). Fluorescence was then measured every 15 min for a period of 14 h. GFP excitation was performed at a wavelength of 488 nm and emission was measured at 522 nm. Relative fluorescence units were calculated by dividing the average fluorescence by the OD600.

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# Team:Humboldt Berlin/Results

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## Results

### Size distribution of produced minicells

We wanted to know whether there is a difference in size of minicells at time points during bacterial cell growth, and how filtration shifts this distribution.

Filtration was done using 0.45 µm filters. The protocol for the conducted measurements can be found [here](https://2021.igem.org/Team%3AHumboldt_Berlin/Experiments).

Comparing density distributions across optical densities for either filtered or not filtered minicells show no significant difference in mean size. Meaning, our measurements show that the time of extraction has no significant influence in the observed time frame. Therefore, we decided to compare all measured cells unfiltered and filtered (using 0.45 µm filter) in one (graphic all\_raw and graphic raw\_filtered).

Filtration or purification on the other hand influences the size distribution substantially, which is only logical. The mean diameter shifted from 529 nm to 394 nm after filtration. If it would be desirable to have minicells of increased size for applications (or not) is further investigated in [Model C](https://2021.igem.org/Team%3AHumboldt_Berlin/Modelling).

Please bear in mind that filtration took a significant hit on the minicell count compared to the non-filtered equivalent. We experienced a loss of up to 7/8 of total minicell count after filtration.

![...](https://static.igem.org/mediawiki/2021/f/fc/T--Humboldt_Berlin--Target_Taxi_fig1.png)

Figure 1: Violin Plots of the experimentally measured size distributions at different growth phases measured in nm. Minicell size was characterized as well in filtered and unfiltered samples (raw).

### Proton Motive Force Measurements

The membrane potential plays a crucial role in maintaining the energy household of cells. Along with the chemical potential, it drives the proton motive force (PMF). The PMF is used in different essential physiological processes, e.g., to produce chemical energy sources such as ATP[1](#Fischer1) and for the fueling of secretory machines, such as the type 3 secretion system of the injectisome[2](#Diepold1). Thus, an important aspect of the minicells that must be characterized is their ability to maintain the membrane potential.

To quantify the time the minicells are able to execute their function we characterized the membrane potential as an indicator for minicell activity. For this, we used DiSC3(5) (3,3'-Dipropylthiadicarbocyanine Iodide), a cationic dye able to accumulate within polarized membranes[3](#Winkel1). By this, the higher the membrane potential the higher the accumulation of DiSC3(5) within the membrane. Therefore, this can be used as an indicator for the functioning of minicells.

We expect an exponential decay of the proton motive force as an indicator for metabolism and function executing minicells. An exponential fit onto the acquired data in the form of I(t) = a \* exp( -c \* t), where I is the intensity, c the reziproke of the lifetime and t the time. This resulted in a = 0.136572 and c = 0.03432747 and can be seen together with the recorded data in figure (PMF). Furthermore we use this acquired lifetime to calculate the lifetime of a minicell dependent on its size in our Model P.

![...](https://static.igem.org/mediawiki/2021/0/0f/T--Humboldt_Berlin--Target_Taxi_fig2.png)

Figure 2: Mean intensity of DiSC3(5) reporter of still gfp expressing minicells, extracted at different time points (black). Exponential fit (blue) with a = 0.136572 and c = 0.03432747.

### Characterization of PlldR (BBa\_K1847008) in *Salmonella*

The lactate-inducible promoter (PlldR) of E. coli is frequently used and well characterized by a variety of iGEM team, highlighted by the high frequency of part-characterization on the respective part web page. Since we are working with *Salmonella* Typhimurium and wanted to use a promoter that is induced by lactate, which is secreted in high amounts by cancer cells. For our project, we therefore decided to characterize the lldR promoter (BBa\_K1847008) in the *S.* Typhimurium background.

For this, we fused PlldR to the green fluorescent protein (*gfp*; BBa\_E0840) reporter gene and cloned it into the pKF01 backbone we designed for our project. pKF01 expresses the lldR gene from a constitutive synthetic promoter (Psyn\_*thyA*, BBa\_K3861002), together with the *thyA* gene (BBa\_K3861003).

We characterized the promoter by using lactic acid at different concentrations. If LldR is not present, PlldR drives expression of *gfp* constitutively, which can be observed in the coloring of the colonies on LB-agar plates. In Figure 1A, the PlldR-*gfp* construct is found in the pSB1C3 backbone, which lacks the repression of LldR. This results in constant expression of *gfp* and thus green colored colonies. On the other hand, when LldR is present in higher amounts, expression from PlldR promoter is restricted/reduced. This can be observed Figure 2B, where PlldR-*gfp* construct was cloned into the pKF01-backbone.

When LldR, the repressor of this promoter, is present in higher amounts, the promoter becomes inducible. Here, LldR binds to the promoter, thus blocking it from being targeted by the RNA-polymerase. Upon presence of an inducer LldR releafs DNA and PlldR is free and transcription is possible. This promoter can be fine-tuned by varying different inducer concentrations (Fig. 3). The use of lactic acid for induction of the PlldR was shown to be detrimental to bacterial growth at higher concentrations, as *S.* Typhimurium strains were able to grow at lactic acid concentration up to 12.5 mM. At higher concentrations (25 and 50 mM lactic acid) growth was inhibited (Fig. 4).

![](https://static.igem.org/mediawiki/2021/1/1f/T--Humboldt_Berlin--Target_Taxi_fig3.png)

Fig. 3: *Salmonella* Typhimurium strains either transformed with pSB1C3-PlldR-gfp (on top) or with pKF01-PlldR-*gfp* (at the bottom) and streaked on either an LB-chloramphenicol or an LB plate. (On Top) In this strain lldR is not additionally expressed from the vector, whereby GFP is constitutively expressed from PlldR, resulting in green colored colonies. (At the bottom) lldR is constitutively expressed on pKF01, which allows for repression of *gfp* expression from PlldR promoter, resulting in characteristic *Salmonella* colony color.

![...](https://static.igem.org/mediawiki/2021/7/71/T--Humboldt_Berlin--dose_dependent.png)

Fig. 4: Dose-dependent activity of PlldR -*gfp* at low lactic acid concentrations (6h post induction). Mean relative fluorescence units (RFU) of GFP using lactic acid concentrations between 0 and 6.25 mM.

![...](https://static.igem.org/mediawiki/2021/4/43/T--Humboldt_Berlin--Target_Taxi_fig4.png)

Fig. 5: *Salmonella* Typhimurium growth curve under different lactic acid concentrations. The optical density at a wavelength of 600 nm was measured for a period of 14 h. Growth was measured at different lactic acid concentrations (0-50 mM).

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**List of Sources**

1. Fischer, *S.* , Gräber, P. & Turina, P. The activity of the ATP synthase from Escherichia coli is regulated by the transmembrane proton motive force. J. Biol. Chem. (2000) doi:10.1074/jbc.275.39.30157.
2. Diepold, A. & Armitage, J. P. Type III secretion systems: The bacterial flagellum and the injectisome. Philosophical Transactions of the Royal Society B: Biological Sciences (2015) doi:10.1098/rstb.2015.0020.
3. te Winkel, J. D., Gray, D. A., Seistrup, K. H., Hamoen, L. W. & Strahl, H. Analysis of antimicrobial-triggered membrane depolarization using voltage sensitive dyes. Front. Cell Dev. Biol. (2016) doi:10.3389/fcell.2016.00029.