Collaboration

Optimization

Introduction

Minicells are 100-500 nm bacterially-derived nanoparticulate products of aberrant binary fission. Since these nanoparticles are bioactive and capable of biosynthesis yet lacking chromosome and considered to be more biosafe than their counterparts bacteria, our main team decided to use minicells for the biosynthesis of a pigment known as indigo.

On the other hand, the lack of the chromosome comes with few disadvantages as well. Since minicells lack a genome they are unable to re-synthesize the transcriptional machinery and endogenously produced co-factors involved in the molecular biological dogma. This, in turn means that minicells suffer from short lifespan as well as lack of ATP to regulate multiple cellular processes and movement.

Therefore, we set out to produce a system in which minicells could generate ATP as well as engage in light-directed movement. We believe that succeeding to create such a system not only possibly could improve pigment yield of our main team, but also prolong minicells life and make them a potential novel nano-based delivery system useful in cell to cell communication, drug administration and even genome engineering.

So, without any further ado let us present you Minisails.

Previous tries and fails

Over the years, major advances have been made in metabolic engineering research. Increased knowledge of metabolic pathways and emerging synthetic biology tools allowed us to build more complex circuits and microorganisms with novel properties. However, using microorganisms to produce various compounds poses a great metabolic burden on microorganisms themselves by depleting ATP (1) . This has caused researchers to find new ways of introducing light - conversion systems in non - photosynthetic organisms. Regarding minicels, a study incorporated a whole glycolysis pathway composed of 7 genes into what he called chromosome free cells. Yet, the system seemed to be functional for only 10 days (2). Another possible way of producing ATP includes the usage of chlorophyll or carotenoid based systems. Yet, the main disadvantage of such systems is their high complexity and thus not suitable for ATP production in minicels.

Proteorhodopsin - a viable solution

What is proteorhodopsin (PR) and why we are interested in it?
Proteorhodopsin (later PR) is a light-activated retinal-containing proton pump that is capable of generating a proton motive force (pmf) across the cell membrane. Without a doubt, proton motive force is essential for multiple processes occurring in the cell including ATP synthesis. First time proteorhodopsin was identified in marine bacteria. There are two major groups of proteorhodopsins : blue and green light absorbing. For our project, we decided to use green light absorbing proteorhodopsin since they are better characterized then the blue light ones.

The action mechanism of proteorhodopsin
Protheorodopsin is a transmembrane protein containing the retinal as its chromophore. Upon the application of light, the retinal gets photo isomerized. This changes the conformational change of proteorhodopsin needed for pumping protons. Proteorhodopsin is an outward pump, meaning it pumps H+ out of the cell into transmembrane space. This results in a proton gradient across the membrane and creates a proton motive force that can be used for ATP generation upon H+ entrance via ATP synthases.

Picture from (3)

Our system

Transforming a plasmid into bacteria that

Overexpresses FtsZ (to produce minicells)
Expresses PR

Inducing the system and finding optimal conditions
Measuring ATP production

Building and transforming plasmid into bacteria

For PR, we received a plasmid from Prof. Paul A. Davison of Sheffield University with the PR gene under the control of the pBAD promoter. This is a tightly regulated promoter.

To overexpress FtsZ, we first needed to clone it out of the NEB E. coli LacIq/LysY (C3013) genome and insert it into pACYCDuet1 plasmid. We chose pACYCDuet1 since it has a T7 promoter which is known for high efficiency. To do that we designed different primers and used CPEC (Circular Polymerase Extension Cloning) as our cloning method. We chose CPEC since it is highly efficient, accurate and easy to use. It doesn’t require restriction, digestion, ligation or single stranded homologous recombination. Moreover, CPEC is relatively fast and can be complicated in as short an amount of time as 15 min for simple inserts (4).

FInally, we chose to double transform both plasmids into NEB C3013 strain since it contains genomically integrated T7 polymerase, and can be induced to express the polymerase through IPTG induction. Protocols for preparing chemically competent cells and transforming can be found at Notebook and Protocols tab under Lab Constructs.
Plasmid Schemes

Inducing the system and finding optimal condition
In our system PR is controlled under pBAD promoter. After carrying literature review we found that the most optimal condition to induce pBAD promoter is to use 0.1% arabinose at the OD600 being equal to 0.1.
FtsZ on the other hand is controlled by T7 promoter on a pACYC-Duet1 backbone. To induce FtsZ, we found that it is best to use 0.4mM IPTG at OD600 0.4 - 0.6 and let it incubate for two hours.
Since PR is a retinal containing proton-pump, the retinal is essential for its proper functioning. Yet, E. coli are not able to natively produce it. While one of the ways, to solve it could be adding an additional genetic circuit for heterologous production of retinal, we decided it the complicated genetic circuit might put an additional burden on minicells and decided to instead supply them with all-trans retinal. From the literature review, we concluded that the most optimal concentration for proper functioning PR is 10uM added at OD600 0.25 - 0.3.

Finally, it has been observed that(5) PR only gets activated when there is a significant depletion of ATP and a decrease in pmf. Yet, E. coli is extremely efficient at managing its resources. As a result, it is very hard to deplete ATP in E. coli unless aerobic respiration is disrupted by adding a respiratory poison. To deplete ATP in E. coli we decided to use sodium azide. While after reading the literature, it seemed that 30mM of azide is an optimal amount, we decided to try different concentrations of the respiratory poison to compare ATP production efficiency by PR at different levels of its depletion. The concentrations we wanted to test were 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 50mM and 60mM. We performed experiments with sodium azide in mother cells for several reasons such as 1) confirming our PR is functional, 2)characterizing PR activity in mother cells with and without azide, 3) confirming optimal concentration of sodium azide for highest ATP production, 4) mimicking the conditions we hope to encounter in minicells.

In our project, we hypothesize, that since minicells don’t contain chromosomes as well as they can’t resynthesize multiple compounds, they are rather inefficient at storing energy and ATP is quickly depleted, resulting in actively H+ pumping PR without the addition of poisonous substances. Measuring ATP
To measure ATP production in cells we will use BacTiter-Glo assay by Promega. The action mechanism is based on luciferase. Cells are lysed and the ATP that was contained in cells, together with oxygen and Magnesium ions oxidizes luciferase to oxyluciferin. During this process, light is produced and light intensity is measured using a plate reader. In return, light intensity is used to determine intracellular levels of ATP.
Experimental conditions at which we will measure ATP are summarized below.

Definitions:

FtsZ pBAD - PR: C3013 strain that has both plasmids for minicell and PR production

FtsZ: C3013 strain that produces minicells but doesn’t produce PR

pBAD - PR: C3013 strain that produces PR but doesn’t produce minicells (testing PR in mother cells)

WT: wild type of C3013 strain

Ret - retinal

NaN3 - azide

Ara - arabinose

Measurements of ATP will be performed at times 0, 5, 10, 15, 20, 25, 30, 40, 50. 60 minutes after adding azide and illuminating cells with light as well as having control groups which will be kept in the dark. We are interested in measuring the cells with azide in PR that have been illuminated and comparing them to cells with azide in PR that were kept completely in the dark. In addition, we will be repeating same experiments with cells without azide. ATP differences measured using BacTiter-Glo between cells kept in the dark and illuminated cells will allow us to make conclusions on PR efficiency.

Additionally, we aim to characterize how long minicells can survive before they deplete all their ATP resources. For this, we will measure the levels of ATP in minicells (FtsZ + pBAD - PR, no azide, only iPTG, arabinose and retinal) kept in the dark at different time intervals, until ATP will approach zero.

Finally, it was noticed (5) that PR in minicells increase their movement speed. To observe minicells movement, we will express mCherry in minicells and use microscopy while illuminating cells with green light ( ~520 nm).
To perform some of the experiments, we needed to purify minicells. For minicell purification, we will the protocol in Notebook & Protocols tab under Lab Constructs. Unfortunately, due to time constraints and issues of proper plasmid cloning and transformation, we didn’t have time to carry out our system induction and ATP measurements.

Future Perspectives and prospects

Minicells due to their features (small size, chromosome-free, bacteria-derived membrane, metabolic activities, ability to carry and express plasmid, etc) have a great potential to become highly efficient nanoparticle cargo carriers. In fact, minicells are already gaining more and more attention in research regarding target drug administration. Keeping that in mind, and knowing that PR is able to power flagellar motors, we would like to further continue working on minicells for directed movement production. Recently, a paper has been published on producing and characterizing motile and chemotactic minicells (6) . Not only they managed to demonstrate that minicells are well-motile but also that they can be chemotactic. Furthermore, they found that minicell chemotactic efficiency is comparable to that of regular bacteria. We would like to further continue and improve their work by using PR to alleviate minicell movement and use the green light needed for PR activation as a tool to create light-directed movement. We hope to demonstrate that minicells can perform phototaxis and further develop tools to control this movement.

Methods

Plasmid Repression

There are numerous methods for expression control of genetic elements on plasmids. These range from differentially inducible promoters that generally only allow expression of downstream genes in the presence of a chemical inducer, as well as toggle switch systems that take advantage of promoter directionality to tightly control the expression of a system of genes (7) . These systems allow engineered microbes to produce recombinant proteins on command, and limit the expression of toxic proteins to survivable levels for the microorganism as well as limiting resource utilization.

For minicells to be viable as a microbial protein production platform, tight control of genetic systems that are present in the mother cell is desirable. However, since mother cells and minicells are similar in their chemical composition (8) , the two ways in which this might be achieved are either:

1. Differential environments

Initially, our project had the aim of not only producing ATP through PR, but also to use the PR to drive directed minicellular motion. If minicells and mother cells are able to autonomously separate and prefer different environments, then plasmids in the mother cell that are induced by the environment preferred by the minicells are repressed. Due to time constraints, we were not able to explore this further but presents an interesting avenue for later research.

2. Genomic plasmid repression

The largest and most apparent difference between minicells and mother cells, are that minicells lack the genome of the cell from which they are derived. This is because minicell development and bacterial division are separate events and do not interfere with one another (9). In this way, a plasmid could be repressed in the presence of the genome and expressed in its absence. For choosing a suitable protein, there are three considerations:

Promoter strength and inducibility.

This affects the quantity and timing of the repressor protein in the cytosol of the microbe. In our system, we decided that the genomic gene should be controlled by a weak constitutive promoter. This will allow for there to be enough protein in the cytosol for repression, but not so much that it is overabundant

Protein half-life in vivo.
Choice of the repressive proteins’ half-life will allow for temporal control of expression in the minicell. For proteins with a long half-life, the delay between minicell formation and minicell protein production would be long, and the opposite for proteins with short half-life. In our design, we did not consider this but would choose a genomic protein with a short half-life. This will allow for relatively rapid expression of recombinant protein post minicell formation.

Due to these two considerations we chose to proceed with the LysY gene, which codes for the protein lysozyme. Lysozyme can bind to T7Pol and inhibit any protein expression under a T7 promoter (10). For this we used the NEB E. coli LacIq\LysY strain (C3013) which has a single copy number MiniF plasmid containing LysY as well as genomically integrated T7Pol under the control of the lac operon, allowing its expression in the presence of IPTG. LysY is downstream of an uncharacterized weak constitutive promoter and thus would be perfect for our application. The plasmid constructions were as illustrated below:

Construction of plasmids

Schematics of constructed and proposed plasmids

Table of constructed plasmids

pACYC-T7-GFP

The tet promoter and GFP coding sequence was isolated from the PCR of an internal plasmid and inserted into the pACYC-Duet plasmid through CPEC and the T7Pol coding sequence was isolated from a PCR of C3013 genomic DNA and inserted through CPEC. Unfortunately, both the insertion of tetP and T7Pol are presumed to have failed due to a lack of validation from colony PCRs. tetP systems are well known for being leaky systems for inducible expression(11) , meaning that they promote the expression of downstream genes without inducer molecules being present. This would have allowed a background level of T7Pol expression in the mother cell.

pLac-FtsZ

pLac-FtsZ stems from a pCOLA-Duet1 plasmid and is used to overexpress FtsZ in order to develop minicells. The plasmid was linearized to remove both T7 promoter sites and was instead substituted with a Plac promoter. FtsZ was isolated from the genomic DNA of C3013 and inserted downstream of the lac promoter using CPEC. Upon addition of IPTG or lactose, this plasmid should start to overproduce FtsZ and form minicells.

MiniF-LysY

The MiniF-LysY plasmid was isolated from C3013. It is a single copy number F plasmid with the ability to produce lysozyme from an uncharacterized constitutive promoter. This would generate a relatively high level of lysozyme in the cytosol of our bacteria, inhibiting any downstream expression from the leaky T7Pol production.

The MiniF-LysY plasmid was isolated through miniprep. MiniF-LysY, pACYC-T7Pol-GFP & pLac-FtsZ would have been transformed into competent NEB E. coli Turbo (C2984). This is because C2984 does not have genomically produced T7Pol and would therefore not express the polymerase upon the addition of IPTG.

In the mother cell, there would be a sustained production of lysozyme which is able to bind to the T7Pol, inhibiting its polymerase activity. Upon FtsZ expression and subsequent minicell formation, T7Pol concentrations should continue to rise slowly, eventually overpowering the In the mother cell, there would be a sustained production of lysozyme which is able to bind to the T7Pol, inhibiting its polymerase activity. Upon FtsZ expression and subsequent minicell formation, T7Pol concentrations should continue to rise slowly, eventually overpowering the remaining lysozyme at which point it should start to synthesize GFP.

GFP production in the minicell. In a minicell, GFP should be produced after a short amount of time. Minicell formation is followed by an increase in minicellular T7Pol as well as a decrease in Lysozyme due to degradation. This lysozyme is not resynthesized and will thus theoretically reach a point where T7Pol is able to overcome its repression and synthesize GFP.

Unfortunately, due to time constraints and failed insertion, the pACYC-T7Pol-GFP plasmid was never fully constructed and our design could not be validated. Below are some schematics of the theoretical interactions between the MiniF-LysY and pACYC-T7Pol-GFP plasmids. pLac-FtsZ is omitted but is activated by the induction of FtsZ.

Simple schematics of proposed and constructed plasmids as well as their interactions

Without IPTG induction, FtsZ is not overproduced and minicells are not developed by the mother cell. In the mother cell the MiniF plasmid interferes with T7Pol activity and the circuit is thus turned off

In the presence of IPTG, FtsZ is overproduced and minicells are developed. In the MiniF-lacking minicell, the T7Pol is able to regain its foothold and begin its synthesis of GFP and the circuit turns on

More information on the plasmids, primers and methodology can be found on our benchling.

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REFERENCES

(1) Tikh, Ilya, and Claudia Schmidt-Dannert. “Chapter 16 - Towards Engineered Light–Energy Conversion in Nonphotosynthetic Microorganisms.” Synthetic Biology, edited by Huimin Zhao, Academic Press, 2013, pp. 303–16. ScienceDirect

(2) Fan, Catherine, et al. “Chromosome-Free Bacterial Cells Are Safe and Programmable Platforms for Synthetic Biology.” Proceedings of the National Academy of Sciences, vol. 117, no. 12, Mar. 2020, pp. 6752–61.

(3) DOI: 10.13140/RG.2.1.1340.5608

(4) Englert, Derek L., et al. “Investigation of Bacterial Chemotaxis in Flow-Based Microfluidic Devices.” Nature Protocols, vol. 5, no. 5, May 2010, pp. 864–72. www.nature.com, https://doi.org/10.1038/nprot.2010.18

(5) Walter, J. M., et al. “Light-Powering Escherichia Coli with Proteorhodopsin.” Proceedings of the National Academy of Sciences, vol. 104, no. 7, Feb. 2007, pp. 2408–12. DOI.org (Crossref), https://doi.org/10.1073/pnas.0611035104.

(6) Ni, Bin, et al. “Production and Characterization of Motile and Chemotactic Bacterial Minicells.” ACS Synthetic Biology, vol. 10, no. 6, June 2021, pp. 1284–91. ACS Publications, https://doi.org/10.1021/acssynbio.1c00012

(7) Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in microbiology, 5, 172.

(8) SHOHAYEB, M., & CHOPRA, I. (1985). Composition of membranes from whole cells and minicells of Bacillus subtilis. Microbiology, 131(2), 345-354.

(9) Adler, H. I., Fisher, W. D., Cohen, A., & Hardigree, A. A. (1967). Miniature Escherichia coli cells deficient in DNA. Proceedings of the National Academy of Sciences of the United States of America, 57(2), 321.

(10) Moffatt, B. A., & Studier, F. W. (1987). T7 lysozyme inhibits transcription by T7 RNA polymerase. Cell, 49(2), 221-227.

(11) Berens, C., & Hillen, W. (2003). Gene regulation by tetracyclines: Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes. European journal of biochemistry, 270(15), 3109-3121.