iGEM 2023 — Team uzurich

{% extends "layout.html" %}
{% block title %} Notebook {% endblock %}
{% block icon %} ![Notebook Icon](https://static.igem.wiki/teams/4662/wiki/icons/book.png) {% endblock %}
{% block lead %} Walking with Us Along Our Project Timeline {% endblock %}
{% block page\_content %}

The following section gives an overview of our progress in the wet lab.
Specific protocols can be found under [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27Experiments%27%29%20%7D%7D).

In the beginning Tamino and Elia were designing our Quorum sensing and crispr system, while starting with performing and planning first experiments.
From July on experiments related to bacteria growth were performed in the seeger lab by Arianna and Tamino, while Julia, Laetitia and Elia completed all the different assemblies.
In the last months, the results were measured by Tamino with the help of Elia and Julia in the Zamboni Lab.

---

## Timeline

### Week: 26.06 - 02.07

After an introduction into the lab and the different equipment, the first vaginal fluid simulant (VFS) was mixed and the pH got adjusted to 4.5 and 6.5..
After autoclaving the medium still contained some debris and changed color.

![](https://static.igem.wiki/teams/4662/wiki/notebook/notebook-1.jpg)

### Week: 26.06 - 02.07

After familiarization with the golden gate assembly methods, we started designing the plasmids for the quorum sensing system (QS).
The QS component parts were found in the iGEM part registry, and the sgRNA parts were designed based on gene target suggestions from dry lab.
A more efficient lactate dehydrogenase (from Streptococcus bovis) was chosen to increase lactate output.

### Week: 03.07 - 09.07

Inoculation of the VFS with *E.coli* and *L. crispatus*.
Since the bacteria didn't grow, the VFS was prepared new and the pH got adjusted.
First experiments with *E.coli* and *L. crispatus* with both pH and aerobic and anaerobic conditions.

### Week: 03.07 - 09.07

All the parts for the QS were ordered from IDT and we continuously started with the design of the dCas9 system.

### Week: 10.07 - 16.07

Further experiments were performed to find the best agar plate for isolated colonies.

![](https://static.igem.wiki/teams/4662/wiki/notebook/notebook-2.jpg)

### Week: 10.07 - 16.07

The sgRNA were designed with the online tool chopchop and the RBS were designed with Sally's LAB RBS calculator.

### Week: 17.07 - 23.07

A new medium simulating vaginal fluid (MSVF) was tested but after autoclaving there were some solid parts inside.

### Week: 17.07 - 23.07

Transformation of level 0 parts and backbones from the iGEM kit and preparing glycerol stocks.
Trying out different versions of mini prep to increase yield and measuring the yield with Nanodrop and Qubit-Kit.

![](https://static.igem.wiki/teams/4662/wiki/notebook/notebook-3.jpg)

### Week: 24.07 - 30.07

The IDT parts for the QS arrived and were diluted with nuclease free water to have a stock solution and a working solution.

The first assembly of four different Level 1 plasmids was mixed, followed by the short incubation protocol.
After the transformation only two assemblies worked.
Therefore, the parts were mixed again and then incubated with the long incubation protocol overnight.
Transformation, overnight culture, and mini prep of Level 1 and sending for sequencing, with unfortunately only one positive result.
Design and ordering adapters for Level 2 assembly of the QS.
The design of the dCas9 system was done and the parts were ordered from IDT.

### Week: 31.07 - 06.08

The medium simulating vaginal fluid (MSVF) was tested again with success.
Growth experiments were performed with different pH and air conditions.

![](https://static.igem.wiki/teams/4662/wiki/notebook/notebook-5.jpg)

### Week: 31.07 - 06.08

Trying first Level 2 assembly.
Preparing *E.coli MG1655* competent cells and transforming further iGEM kit parts.
Competent cells containing the plasmid pfd152, which contains the dCas9 enzyme, were made and aliquoted for further use.

![](https://static.igem.wiki/teams/4662/wiki/notebook/notebook-4.jpg)

### Week: 07.08 - 13.08

Growth curves experiments were being planned.
An overnight culture will be subcultured in the morning and then every two hours the OD will be measured.
For platting the bacteria, a dilution series in H20: Peptone will be made and then 10ul is placed on the plate.

### Week: 07.08 - 13.08

First positive sequencing results and further trying Level 1 assembly since not all of them worked.
After not having success with the transformation of the backbone jump47-ts for weeks, it was being realized that the incubation temperature should have been 30°C and not 37°C.
After switching the temperature, the transformation worked but the yield after the mini prep was still too low, even with trying a different protocol for the overnight culture.

### Week: 14.08 - 20.08

MRS and LAPTg media, as well as LAPTg plates were prepared.
The MRS media was being tested.
Further preparations for the growth curves were done and a first test run.

### Week: 14.08 - 20.08

Since there were still two level 1 plasmid missing a 2:1 assembly ration of the inserts and backbone was being tested, and the promoter was switched to a different Anderson promoter.
The assembly with a new promoter and 1:1 ration worked out great and the assembly was confirmed with a positive sequencing result.
PCR and gel purification for changing the overhangs of LDH for chromosomal integration with pOSIP-TT, amplification of the dCas9 out of the pfd152 plasmid and homology arms out of the bacterial genome.

![](https://static.igem.wiki/teams/4662/wiki/notebook/notebook-6.jpg)

### Week: 21.08 - 27.08

First growth curves experiment with *E.coli* and *L.crispatus* in LAPTg and LB liquid medium and a dilution series for plating on both plates.

![](https://static.igem.wiki/teams/4662/wiki/notebook/notebook-7.jpg)

### Week: 21.08 - 27.08

Since only the PCR worked for one pair of the homology arms, several different versions for the PCR and different agarose percentage for the gel were tested.
In the meantime, the parts from IDT arrived.
With all the sgRNA we did a PCR to anneal the promoter and change the overhangs.
Unfortunately, we realized that the PCR fragments were too small to do Gel purification with the kit.

Trying Level 2 QS assembly and upstream assembly in the jump27-ts backbone without success.
Transformation of both pOSIP-plasmids from the kit but only pOSIP-CH worked.

### Week: 29.08 - 03.09

Ordering new sgRNA fragments where the promoter and scaffold is already attached and dCas9 enzyme with a degradation tag from the Twist.
In the meantime, we tried Ethanol purification for PCR sgRNA products.
It worked for one sample, but the sequencing showed many mutations in the gRNA.

Trying Level 2 assembly without success.

### Week: 04.09 - 10.09

Arrival of new sgRNA and assembly with great success.
Making agar plates for blue- white screen and trying screen.
No colonies grew.

![](https://static.igem.wiki/teams/4662/wiki/notebook/notebook-8.jpg)

### Week: 11.09 - 17.09

Growth curves with *L. gasserei* and *L. crispatus* in MRS.

### Week: 11.09 - 17.09

PCR with new design primers for LDH assembly to change the overhang for pOSIP- CH chromosomal integration.
After the sequencing we tried to integrate it, which did not work out.
The five different RBS level 1 plasmids were assembled and transformed.

![](https://static.igem.wiki/teams/4662/wiki/notebook/notebook-9.jpg)

### Week: 18.09 - 24.09

Growth curves with *L. crispatus* and *E.coli* in co-culture.
An overnight culture for each of the species and one containing both was made, and the next day the same procedure as for all growth curves experiments.
Each culture was being platted on COS and LB after diluting in H20: Peptone.

![](https://static.igem.wiki/teams/4662/wiki/notebook/notebook-10.jpg)

### Week: 18.09 - 24.09

After weeks of unsuccessful Level 2 assembly for the QS, we realized that the backbones in the Level 1 assembly were wrong.
The Level 1 assembly was mixed new, transformed, followed by a positive sequencing result.
Agar plates for the blue white screen were prepared by the media kitchen.
Trying a different version to plate the LacZ sgRNA in the pfd152 competent cells.
No success in inducing the expression of the dCas9.
Only blue colonies.

![](https://static.igem.wiki/teams/4662/wiki/notebook/notebook-11.jpg)

### Week: 25.09 - 01.10

With the new Level 1 assembly for the QS, we started with the Level 2 assembly.
Preparation of RhlI level 1 plasmid to test with metabolomics.
The sgRNA were transformed in the competent cells containing the dCas9.
Overnight cultures were inoculated, and the first measurements with the D-lactic kit were made.

### Week: 02.10 - 08.10

Visualization of growth curves.

### Week: 02.10 - 08.10

Since the first try for the Level 2 QS plasmids was unsuccessful, the experiment was being performed again.
This time with success.
Further measurements of the efficiency of the sgRNA and first measurements of the RhlR + CFP assembly and the RhlI Level 1.
Also testing the RhlI samples.

![](https://static.igem.wiki/teams/4662/wiki/notebook/notebook-12.jpg)

### Week: 9.10 - 12.10

First measurement of the lactate dehydrogenase from the *Streptococcus bovis* in the knockout strain.
Testing the expression of the LDH with constitutive promoter expression, inducing it manually by adding C4-Hsl to the RhlR + LDH Level 2.
Testing the self-induction of the Level 2 RhlR + CFP construct with co-transformation of RhlI, which produces the auto-inducer molecule.

{% endblock %}

{% set introduction\_sources = [
{"names": "[1.] Fayol-Messaoudi D, Berger CN, Coconnier-Polter MH, Liévin-Le Moal V, Servin AL.", "title": "pH-, Lactic acid-, and non-lactic acid-dependent activities of probiotic Lactobacilli against Salmonella enterica Serovar Typhimurium (2005).", "link": "https://pubmed.ncbi.nlm.nih.gov/16204515/"},
{"names": "[2.] Alakomi HL, Skyttä E, Saarela M, Mattila-Sandholm T, Latva-Kala K, Helander IM.", "title": "Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane (2000).", "link": "https://pubmed.ncbi.nlm.nih.gov/10788373/"},
{"names": "[3.] Wang C, Chang T, Yang H, Cui M.", "title": "Antibacterial mechanism of lactic acid on physiological and morphological properties of Salmonella Enteritidis, Escherichia coli and Listeria monocytogenes (2015).", "link": "https://doi.org/10.1016/j.foodcont.2014.06.034"},
{"names": "[4.] Ricke SC.", "title": "Perspectives on the use of organic acids and short chain fatty acids as antimicrobials (2003).", "link": "https://doi.org/10.1016/j.foodcont.2014.06.034"},
{"names": "[5.] Farhana A, Lappin SL.", "title": "Biochemistry, Lactate Dehydrogenase (2023).", "link": "https://www.ncbi.nlm.nih.gov/books/NBK557536/"},
] %}
{% set considerations\_sources = [
{"names": "[1.] Wikipedia.", "title": "Flux.", "link": "https://en.wikipedia.org/wiki/Flux\_(metabolism)#:~:text=Flux%2C%20or%20metabolic%20flux%20is,pathway's%20activity%20under%20different%20conditions"},
{"names": "[2.] Wikipedia.", "title": "Quorum Sensing.", "link": "https://en.wikipedia.org/wiki/Quorum\_sensing"},
{"names": "[3.] Wikipedia.", "title": "Golden Gate Cloning.", "link": "https://en.wikipedia.org/wiki/Golden\_Gate\_Cloning"},
{"names": "[4.] Valenzuela-Ortega M, French C.", "title": "Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology (2021).", "link": "https://doi.org/10.1093/synbio/ysab003"},
] %}
{% set qs\_sources = [
{"names": "[1.] Team Tokyo Tech.", "title": "iGEM Wiki (2014).", "link": "https://2014.igem.org/Team:Tokyo\_Tech"},
{"names": "[2.] .", "title": "Bacterial Expression Support", "link": "https://www.thermofisher.com/ch/en/home/technical-resources/technical-reference-library/protein-expression-support-center/bacterial-expression-support/bacterial-expression-support-getting-started.html"},
{"names": "[3.] Kumar L, Patel SKS, Kharga K, Kumar R, Kumar P, Pandohee J, Kulshresha S, Harjai K, Chhibber S.", "title": "Molecular Mechanisms and Applications of N-Acyl Homoserine Lactone-Mediated Quorum Sensing in Bacteria (2022).", "link": "https://www.mdpi.com/1420-3049/27/21/7584"},
{"names": "[4.] iGEM.", "title": "Part BBa\_R0071", "link": "https://www.thermofisher.com/ch/en/home/technical-resources/technical-reference-library/protein-expression-support-center/bacterial-expression-support/bacterial-expression-support-getting-started.html"},
] %}
{% set dehydrogenase\_sources = [
{"names": "[1.] Mazumdar S, Blankschien MD, Clomburg JM et al.", "title": "Efficient synthesis of L-lactic acid from glycerol by metabolically engineered Escherichia coli.", "link": "https://doi.org/10.1186/1475-2859-12-7"},
] %}
{% set dcas\_sources = [
{"names": "[1.] Tülay Aşkin Çelik.", "title": "Cytotoxicity (2017).", "link": "https://www.intechopen.com/chapters/61438"},
{"names": "[2.] Depardieu F, Bikard D.", "title": "Gene silencing with CRISPRi in bacteria and optimization of dCas9 expression levels (2019).", "link": "https://pubmed.ncbi.nlm.nih.gov/31377338/"},
{"names": "[3.] Zhang R, Xu W, et al.", "title": "Gene Silencing Through CRISPR Interference in Bacteria: Current Advances and Future Prospects (2021).", "link": "https://www.frontiersin.org/articles/10.3389/fmicb.2021.635227/full"},
{"names": "[4.] (Salislab).", "title": "RBS Calculator", "link": "https://salislab.net/software/predict\_rbs\_calculator"},
{"names": "[5.] Depardieu F, Bikard D.", "title": "Derivative of pFD116 with an optimized RBS for E. coli", "link": "https://www.addgene.org/125546/"},
] %}
{% set sgrna\_sources = [
{"names": "[1.] Chang DE, Jung HC, Rhee JS, Pan JG.", "title": "Homofermentative production of D- or L-lactate in metabolically engineered Escherichia coli RR1 (1999).", "link": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC91196/"},
{"names": "[2.] Kochhar S, Hottinger H, Chuard N, Taylor PG, Atkinson T, Scawen MD, Nicholls DJ.", "title": "Cloning and overexpression of Lactobacillus helveticus D-lactate dehydrogenase gene in Escherichia coli (1992).", "link": "https://pubmed.ncbi.nlm.nih.gov/1396685/"},
{"names": "[3.] Taguchi H, Ohta T.", "title": "D-lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family. Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum (1991).", "link": "https://pubmed.ncbi.nlm.nih.gov/1840590/"},
{"names": "[4.] Zhu Y, Eiteman M, DeWitt K, Altman E.", "title": "Homolactate fermentation by metabolically engineered Escherichia coli strains (2007).", "link": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1796981/"},
{"names": "[5.] Dien BS, Nichols NN, Bothast RJ.", "title": "Recombinant Escherichia coli engineered for production of L-lactic acid from hexose and pentose sugars (2001).", "link": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1796981/"},
{"names": "[6.] Tian K, Chen Z, Shen W.", "title": "High-efficiency conversion of glycerol to D-lactic acid with metabolically engineered Escherichia coli (2012).", "link": "https://www.ajol.info/index.php/ajb/article/view/101608/90809"},
{"names": "[7.] Wang Y, Tian T, Zhao J et al.", "title": "Homofermentative production of D-lactic acid from sucrose by a metabolically engineered Escherichia coli (2012).", "link": "https://doi.org/10.1007/s10529-012-1003-7"},
{"names": "[8.] Hong A, Cheng K, Peng F, Zhou S, Sun Y, Liu C, Liu D.", "title": "Strain isolation and optimization of process parameters for bioconversion of glycerol to lactic acid (2019).", "link": "https://doi.org/10.1002/jctb.2209"},
{"names": "[9.] Guest JR.", "title": "Anaerobic growth of Escherichia coli K12 with fumarate as terminal electron acceptor. Genetic studies with menaquinone and fluoroacetate-resistant mutants (1979).", "link": "https://pubmed.ncbi.nlm.nih.gov/393800/"},
{"names": "[10.] Cunningham PR, Clark DP.", "title": "The use of suicide substrates to select mutants of Escherichia coli lacking enzymes of alcohol fermentation (1986).", "link": "https://pubmed.ncbi.nlm.nih.gov/3550385/"},
{"names": "[11.] Gupta S, Clark DP.", "title": "Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation (1989).", "link": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC210107/"},
{"names": "[12.] Zhou S, Causey TB, Hasona A, Shanmugam KT, Ingram LO.", "title": "Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110 (2003).", "link": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC152430/"},
{"names": "[13.] Zhou S, Shanmugam KT, Ingram LO.", "title": "Functional replacement of the Escherichia coli D-(-)-lactate dehydrogenase gene (ldhA) with the L-(+)-lactate dehydrogenase gene (ldhL) from Pediococcus acidilactici (2003).", "link": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC154814/"},
{"names": "[14.] Valenzuela-Ortega M, French C.", "title": "Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology (2021).", "link": "https://doi.org/10.1093/synbio/ysab003"},
{"names": "[15.] Depardieu F, Bikard D.", "title": "Gene silencing with CRISPRi in bacteria and optimization of dCas9 expression levels (2019).", "link": "https://www.addgene.org/125546/"},
{"names": "[16.] ", "title": "D-Lactic Acid (D-Lactate) (Rapid) Assay Kit.", "link": "https://www.megazyme.com/d-lactic-acid-assay-kit"},
] %}
{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block icon %} ![Engineering Icon](https://static.igem.wiki/teams/4662/wiki/icons/benchling.png) {% endblock %}
{% block lead %} **Our *Design → Build → Test → Learn* Cycle** {% endblock %}
{% block page\_content %}

Our *LactoBack* helper bacterium combines a higher-efficiency lactate dehydrogenase (LDH), CRISPRi-mediated flux redirection as well as population density sensing using the quorum sensing (QS) system found in *P. aeruginosa*.
To test our system, we performed *Design-Build-Test-Learn* cycles on four system components: QS, LDH expression, dCas9 expression and sgRNA expression.

All our Designed Parts and Successful Transformation and assembly can be found [here](https://static.igem.wiki/teams/4662/wiki/important/lactoback-engineering.pdf).

## Introduction

We aimed to engineer a bacteria that would restore a “healthy” *Lactobacillus*-dominated vaginal microbiome by secreting more lactic acid which exhibits antimicrobial properties [[1.]](#IntroductionReferences).

![](https://static.igem.wiki/teams/4662/wiki/schematics/helper-bacterium-corrected.png)

Figure 1: Schematic overview of the helper bacterium system circuit.

A quorum sensing mechanism is dependent on population density and leads to a conditional activation of dCAS9 expression and lactate dehydrogenase expression.

This results in flux redirection towards lactate production.

Figure 1 is an illustration of the designed system to elevate lactic acid production in our "helper bacterium".

1. Firstly, we wanted to **increase the amount of lactate dehydrogenase**: an enzyme that catalyzes the reaction of pyruvate to lactic acid [[5.]](#IntroductionReferences).
   This would help to increase lactate production.
2. Secondly, we wanted to **knock down non-essential genes involved in turning pyruvate into substances other than lactic acid using  CRISPRi**.
   By doing so, metabolic flux would be directed into lactic acid production.
3. Finally, we aimed to **couple this system to bacterial communication (quorum sensing - QS)**, so that our bacterium would only secrete more lactic acid when it was doing well - which indicates dysbiotic conditions in the vagina, i.e. conditions that require medication / preventative action.

{% for introduction\_source in introduction\_sources %}

[{{ introduction\_source.names }} *{{ introduction\_source.title }}*](%7B%7B%20introduction_source.link%20%7D%7D)

{% endfor %}

---

## Design Considerations

The overall amount of lactic acid produced is dependent both on the amount produced per bacterium and the number of bacteria.

To maximize production of lactic acid in a single bacterium we planned to overexpress lactic acid dehydrogenase (LDH) and redirect  metabolic flux [[1.]](#ConsiderationsReferences) using  dCas9 and a multiplexing construct containing four  sgRNAs.

However both these methods lower the fitness of the helper bacterium, making it susceptible to being outcompeted by WT bacteria.

We therefore decided to couple the overproduction of lactic acid to a  quorum sensing [[2.]](#ConsiderationsReferences) system.
This enables the bacterial population to grow fast initially, until it reaches a threshold level where expression of additional LDH and dCas9 are induced.
Moreover it maximizes the long term output of lactic acid by ensuring the population is of sufficient size to produce a substantial amount of lactic acid.

The components of this system were assembled in four separate plasmids that could later be chromosomally integrated [(Figure 2)](#Figure2).

Because some of the system's components (dCas9, sgRNAs, see following sections), require the screening of different variations to perform optimally, we decided to assemble our plasmids using  Golden Gate cloning [[3.]](#ConsiderationsReferences) and the Joint universal modular plasmids (JUMP) collection [[4.]](#ConsiderationsReferences).

![](https://static.igem.wiki/teams/4662/wiki/plasmids/prhlr-rhli.gif)

![](https://static.igem.wiki/teams/4662/wiki/plasmids/pldh.gif)

![](https://static.igem.wiki/teams/4662/wiki/plasmids/pmultiplex.gif)

![](https://static.igem.wiki/teams/4662/wiki/plasmids/pdcas9.gif)

Figure 2. Plasmid maps of LactoBack system components:

pRhlR-RhlL contains transcriptional units (TU's) of the QS system.
pRhlI is necessary for the production of the QS C4-HSL and RhlR encodes for the transcription factor that binds C4-HSL and the conditional promoter LasB.
pLDH conditionally expresses LDH.
pdCas9 is responsible for conditional expression of dCas9 in response to the QS system.
pMultiplex contains four constitutively expressed sgRNAs targeting multiple loci.

All vectors were designed using Benchling.
Each of the plasmids was to be constructed and optimized by running an individual DBTL cycle.

{% for considerations\_source in considerations\_sources %}

[{{ considerations\_source.names }} *{{ considerations\_source.title }}*](%7B%7B%20considerations_source.link%20%7D%7D)

{% endfor %}

---

## DBTL Quorum Sensing

#### Design

We chose to show our proof of concept in *E. coli* because it is both well studied and easy to engineer.
However, if our concept would prove to be successful the system could be transferred to different bacterial species naturally present in the vaginal microbiome, such as *L. gasseri* or *L.Crispatus*.
We chose to use the Rhl quorum sensing system from *Pseudomonas aeruginosa*, which is small, allowing for fast diffusion and functions in both gram positive and gram negative bacteria [[1.]](#QSReferences).
We chose to isolate three components of the QS system that can function autonomously.
The molecule (C4-HSL produced by RhlI) and a transcription factor (RhlR) that activate the conditional promoter LasB [[2.]](#QSReferences).

The quorum sensing system should be chromosomally integrated using pOSIP-CH.

#### Build

Assembly of RhlR TU in pJUMP29-1A and RhlI TU in pJUMP29-1B.

The resulting vectors were successfully assembled into the  backbone pJUMP49-2B using a linker to substitute for the missing fragments (Level two plasmids usually contain four TU's).
However, transfer of the resulting plasmid

pRhlR-RhlL
into
 pOSIP-CH failed.
Thus no chromosomal integration was possible.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4662/wiki/plasmids/prhlr-rhli.gif) | | | |
|  | Promoter: BBa\_J23106 | RBS: BBa\_K2680529 | CDS: BBa\_C0171 | Terminator: Bba\_J435371 |
|  | Promoter: BBa\_J23106 | RBS: BBa\_K2680529 | CDS: BBa\_K4662044 | Terminator: Bba\_J435371 |
|  | Backbone: pJUMP49-2B(sfGFP) | | | |

#### Test

Due to the difficulties we faced during chromosomal integration, we tested the functionality of the complete QS system by testing both individual components and the entire system through different co-transformations.

Recombinant production of C4-HSL by RhlI was tested using FIA-MS.

![](https://static.igem.wiki/teams/4662/wiki/results/fia-ms-rhli.png)

Figure 3. Spectrum of FIA-MS measurement:

The intensity is measured at 170 m/z, which corresponds to the deprotonated C4-HSL molecule.

A3 = supernatant of untransformed *E. coli*, B3 = supernatant of engineered *E. coli*, C1 = extracted cell pellet of untransformed bacterium, D1 = extracted cell pellet of engineered *E. coli*

Sensitivity of RhlR and LasB were tested by assembly of a plasmid containing RhlR and CFP (cyan fluorescent protein) under the expression of the QS dependent promoter LasB.

pRhlR-CFP
was manually induced.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4662/wiki/plasmids/prhlr-cfp.gif) | | | |
|  | Promoter: BBa\_J23106 | RBS: BBa\_K2680529 | CDS: BBa\_C0171 | Terminator: Bba\_J435371 |
|  | Promoter: BBa\_R0079 | RBS: BBa\_K2680529 | CDS: BBa\_K4662027 | Terminator: Bba\_J435371 |
|  | Backbone: pJUMP49-2B(sfGFP) | | | |

![](https://static.igem.wiki/teams/4662/wiki/results/lasb-cfp-raw.png)

![](https://static.igem.wiki/teams/4662/wiki/results/lasb-cfp-corr.png)

Figure 4.

Left: Raw measurement of fluorescence. The untransformed E. coli also has a fluorescent signal at 429 nm excitation.

Right: Values corrected for the fluorescent signal of the negative control.

After successfully testing the RhlR +CFP level 2 plasmid by manually induction and the positive RhlI level 1 plasmid expression and diffusion results, we co transformed both constructs into one bacterium.
With the CFP output, we measured the fluorescent signal over time.

![](https://static.igem.wiki/teams/4662/wiki/results/final-plot-induction.png)

Figure 5.

Fluorescent signal at 429 nm excitation of the transformed but uninduced E.coli compared tothe transformed and induced *E.coli*.

Figure 5 confirms that the CFP signal increases as soon as the population density of the bacteria increases. Simultaneously with the production of the C4-HSL molecule.

#### Learn

Fluorescence was higher in induced plasmids confirming the functionality of the QS system.
The however the difference in fluorescence is relatively small compared to background noise.
That led to the conclusion that LasB showed  leaky expression, especially in the exponential phase of the bacteria when they are highly metabolically active.

To increase the sensitivity of the system a different Rhl dependent promoter could be used in the future [[4.],[5.]](#QSReferences).

After complete characterization of our rhl-las quorum sensing hybrid system, we were able to confirm that RhlR is also able to induce the LasB promoter and the CFP moiety can be easily switched to the sequence of the protein of interest.

{% for qs\_source in qs\_sources %}

[{{ qs\_source.names }} *{{ qs\_source.title }}*](%7B%7B%20qs_source.link%20%7D%7D)

{% endfor %}

---

## DBTL Lactate dehydrogenase

#### Design

The endogenous LDH of E. coli we chose to express L-LDH from *Steptococcus bovis*.

It has high efficiency (kcat/Km value) and has previously been used for overproduction of Lactic acid in *Escherichia coli* [[1.]](#DehydrogenaseReferences).
The sequence should be chromosomally integrated using pOSIP-TT.

#### Build

L-LDH under the expression of LasB successful assembly into pJUMP29-1C.
To compare the inducible LDH expression, we assembled a plasmid where the LDH is constitutively expressed pLDHconst, which was also transformed in the knockout strain.

#### Test

To characterize the function of L-LDH under the expression of LasB we assembled a plasmid containing RhlR and LDH.
The plasmid was induced by manually adding C4-HSL and the resulting lactate production was measured.
To screen for minimal induction concentrations, a dilution series of the C4-HSL molecule was performed.

The constitutive expression of LDH was tested.

![](https://static.igem.wiki/teams/4662/wiki/results/l-lactate-induction.png)

Figure 6. Measurement of L-lactate abundance in control strains; untransformed and knock out, as well as constitutive expression. Comparison to induced L-LDH via LasB promotor.

#### Learn

The constitutive promoter expressing LDH did not result in higher production of lactate compared to the control strain.
As a next step, we would have exchanged the promoter for a stronger unit and repeated the experiment to have a better positive control.

As anticipated, the activation of the LasB promoter produced favorable outcomes, consistent with its prior characterization involving the expression of CFP.

{% for dehydrogenase\_source in dehydrogenase\_sources %}

[{{ dehydrogenase\_source.names }} *{{ dehydrogenase\_source.title }}*](%7B%7B%20dehydrogenase_source.link%20%7D%7D)

{% endfor %}

---

## DBTL dCas9

#### Design

Due to the fact that dCas9 shows  cytotoxic [[1.]](#dCasReferences) effect [[2.],[3.]](#dCasReferences) when expressed at a high level we aimed for an dependent expression, which is activated by the LasB promoter.

LasB is much leakier than the Tet promoter used in many dCas9 systems.
To reduce the presence of dCas9 in the uninduced state we decided to add a degradation tag to increase the turnover rate of the protein.

To further optimize the level of dCas9 present in the cell we screened different RBS's we designed using the Salis lab RBS calculator [[4.]](#dCasReferences).
Additionally, we made a RBS library by randomly mutating four bases as described by Bikard et Al. [[1.]](#dCasReferences).

To select the optimal RBS we designed a blue white screen [(Figure 7)](#Figure7).
For this purpose, we constructed an additional plasmid containing RhlR and a sgRNA (pRhlR-LacZ) targeting the LacZ operon.

Expression of dCas9 with variable RBSs (pdCas9) in combination with the LacZ1-3 sgRNA should result in the silencing of the LacZ operon resulting in white colonies on X-Gal plates.
The desired amount of C4-HSL can be added to the system manually to induce the expression of dCas9.
Colonies that appear blue in the absence of C4-HSL (no silencing in uninduced state), and white in presence of C4-HSL (silencing in induced state) carry a plasmid with ideal RBS strength.

![](https://static.igem.wiki/teams/4662/wiki/schematics/blue-white-screening.png)

Figure 7. Schematic representation of blue white screen

See the Assembly Scheme [here](https://static.igem.wiki/teams/4662/wiki/important/crispri-dcas9-assembly-scheme.pdf).

#### Build

**Vector Assembly**

We successfully assembled different versions of

pdCas9
containing rationally designed RBSs and plasmids containing the RBS library.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4662/wiki/plasmids/pdcas9.gif) | | | |
|  | Promoter: BBa\_R0079 | RBS: Variable- BBa\_K4662000 BBa\_K4662001 BBa\_K4662002 BBa\_K4662003 | CDS: BBa\_K4662061 | Terminator: BBa\_K4662004 |
|  | Backbone: pJUMP29-1C(sfGFP) | | | |

#### Test

**Blue white Screen**

LacZ sg RNAs were tested by co transformation with pFD152 a plasmid containing dCas9 under the expression of a Tetracycline dependent promoter [[4.]](#dCasReferences).

Blue white screen was repeated several times using different batches of X-Gal plates and different levels of tetracycline as well as different methods for induction by tetracycline.
However, colonies always appeared blue.

Co-transformation of a plasmid containing LacZ sgRNA and RhlR (

pRhlR-LacZ
) with our QS dependent dCas9 plasmid also failed to produce white colonies.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4662/wiki/plasmids/prhlr-lacz.gif) | | | |
|  | Promoter: BBa\_J23106 | RBS: BBa\_K2680529 | CDS: BBa\_C0171 | Terminator: Bba\_J435371 |
|  | Promoter: BBa\_J23102 | RBS: BBa\_K4662018 | CDS: BBa\_K4662031 | Terminator: BBa\_K4662007 |
|  | Backbone: pJUMP49-2B(sfGFP) | | | |

![](https://static.igem.wiki/teams/4662/wiki/results/lacz-sgrna-induction-unsuccessful.png)

Figure 8. Unsuccessful induction of the LacZ sgRNA to block the beta-galactosidase

#### Learn

Blue white screen of LacZ sgRNAs could have failed due insufficient activation of the Tet promoter that regulates dCas9.
To ensure sufficient activation, anhydrotetracycline which shows a stronger binding affinity and is not toxic to bacteria could be used to induce dCas9 expression.
However it is also likely that the LacZ sgRNAs performed poorly.
This is supported by the fact that co transformation of a plasmid containing LacZ sgRNA and RhlR with our QS dependent dCas9 plasmid also failed to produce white colonies.
In a repeat experiment a different set of sgRNAs should be designed.

{% for dcas\_source in dcas\_sources %}

[{{ dcas\_source.names }} *{{ dcas\_source.title }}*](%7B%7B%20dcas_source.link%20%7D%7D)

{% endfor %}

---

## DBTL sgRNA

### Cycle 1

#### Design

Optimal targets for flux redirecting were determined by dry lab modeling and verified using literature review [[1.]-[13.]](#sgRNAReferences).
The final targets were PflA, PflB and adhE. Additionally, we planned to knock out pta and ackA by chromosomally integrating at the corresponding site [[14.]](#sgRNAReferences).
The sgRNA were designed with the online tool chop chop. The three gRNA were selected to target the promoter or the beginning of the gene, and have a high predicted efficiency [[15.]](#sgRNAReferences).

| # | pyruvate formate-lyase 1-activating enzyme (pflA) | formate acetyltransferase 1 (pflB) | bifunctional aldehyde-alcohol dehydrogenase (adhE) |
| --- | --- | --- | --- |
| 1 | 76.91 | 70.06 | 54.97 |
| 2 | 68.36 | 64.83 | 70.24 |
| 3 | 67.99 | 69.98 | 69.67 |

Table 1. Predicted efficiency by [chopchop](https://chopchop.cbu.uib.no/)

With PCR and our designed primers the gRNA should be adapted so that the promoter and an overhang for scarless assembly with the scaffold will be annealed.

Designed primers for amplification out the homology arms for AckA out of the bacterial genome.

|  | AckA upstream |
| --- | --- |
| Forward | CGTCTCAggagCGTCTTTGAGTAATGCTGTCCC |
| Reverse | CGTCTCAggctGGAAGTACCTATAATTGATACGTGGC |

|  | AckA downstream |
| --- | --- |
| Forward | CGTCTCAttcgTTTCACACCGCCAGCTCAGC |
| Reverse | CGTCTCAagcgCCTTCAACCAGAACGACTTCAGCG |

#### Build

![](https://static.igem.wiki/teams/4662/wiki/results/pcr-sgrna-1.png)

![](https://static.igem.wiki/teams/4662/wiki/results/pcr-sgrna-2.png)

Figure 9. 1.5% agarose gel with 1kb ruler with the sgRNA after PCR

PCR was performed and the product was loaded on a gel, followed by gel purification.
After sending it for sequencing, we realized that our assembled fragment is too short to perform the Gel or PCR purification with the spin column (only for fragments > 50bp).
We tried the PCR again and performed an ethanol purification.
We sent the samples again for sequencing and only one positive result came back.

![](https://static.igem.wiki/teams/4662/wiki/results/sgrna-pcr.png)

Figure 10. Positive sequencing result after PCR and ethanol purification

The bacteria carrying sgRNAs produced more lactic acid in an induced state compared to the uninduced control and the concentration of 5ug/ml tetracycline showed a better induction (the Protocols are [here](%E2%80%9Dhttps%3A//static.igem.wiki/teams/4662/wiki/important/lactoback-protocols.pdf%E2%80%9D)).

Therefore the gRNA were ordered as a whole construct which already contains the promoter, scaffold and terminator.
With the new version all level 1 sgRNA plasmids were successfully assembled and transformed.

#### Test

To test their efficiency, Level 1 sgRNAs were co transformed with pFD152 [[15.]](#sgRNAReferences) and lactate content was measured using Megazyme D-lactic acid assay kit [[16.]](#sgRNAReferences).
The dCas9 tet- promoter was induced with a 0.5ug/ml and 5ug/ml tetracycline concentration.

![](https://static.igem.wiki/teams/4662/wiki/results/lactate-calibration-line-multinduce.png)

![](https://static.igem.wiki/teams/4662/wiki/results/lactate-adhe2-multinduce.png)

Figure 11.

Left) Standard curve for D-lactate measurement.

Right) The lactate production transformed bacterium was measured in three conditions:
“uninduced” as a baseline how much lactate is produced normally and “induced” by either 0.5ug/ml and 5 ug/ml tetracycline.

#### Learn

We learn from our preliminary data that the sgRNA were able to block the enzymes, resulting in a higher lactic acid concentration when the dCas9 was induced with 5mg/ml tetracycline.
Additionally we concluded that higher induction with tetracycline is not toxic for the cells. For future experiments, only the high induction concentration will be used.

We tested the sgRNA level 1 plasmids again.

![](https://static.igem.wiki/teams/4662/wiki/results/level1-sgrna-grid.png)

Figure 12. The figures (a-i) show the corresponding lactate concentration at each timestep, in both conditions (uninduced and induced).

The predicted efficiency which was made at the beginning of the designing, had only a difference of around 10%.
With our results, we are neither able to support nor deny the same efficiency order as the predictions.

### Cycle 2

#### Design

After testing all the level 1 sgRNA constructs, we selected four sgRNA which showed the best effect for our level 2 plasmid.
The following level 1 where chosen:

Based on the results obtained from the individual sgRNA screening we decided to assemble PflA1, PflB1, AdhE1 and AdhE2 into pMultiplex.
For the guide RNAs of PflB and AdhE, there were nearly no differences measured in their activity.
For PflA, the graphs show that the efficiency might be higher for PflA2 or PflA3, however we can see that the standard deviation is quite big compared to the PflA1 guide RNA.
We focused on the most robust data, which also correlated with the prediction made by chopchop that PflA1 is the most efficient guide RNA.
PflB1 was chosen because it has a highest predicted efficiency and its targeting sequencing in the promoter region.
For AdhE we chose AdhE1 with a targeting area in the promoter and AdhE2 with the highest predicted efficiency.

#### Build

pMultiplex
(Level 2 sgRNA) was assembled with

AdhE1
,

AdhE2
,

PflA1
and

PflB2
.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4662/wiki/plasmids/pmultiplex.gif) | | | |
|  | Promoter: BBa\_J23118 | crRNA: BBa\_K4662021 | tracrRNA: BBa\_C0171 | Terminator: BBa\_K4662010 |
|  | Promoter: BBa\_J23118 | crRNA: BBa\_K4662022 | tracrRNA: BBa\_K4662031 | Terminator: BBa\_K4662010 |
|  | Promoter: BBa\_J23119 | crRNA: BBa\_K4662012 | tracrRNA: BBa\_K4662031 | Terminator: BBa\_K4662008 |
|  | Promoter: BBa\_J23100 | crRNA: BBa\_K4662014 | tracrRNA: BBa\_K4662031 | Terminator: BBa\_K4662009 |
|  | Backbone: pJUMP49-2B(sfGFP) | | | |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4662/wiki/plasmids/padhe1.gif) | | | |
|  | Promoter: BBa\_J23118 | crRNA: BBa\_K4662021 | tracrRNA: BBa\_K4662031 | Terminator: BBa\_K4662010 |
|  | Backbone: pJUMP29-1D(sfGFP) | | | |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4662/wiki/plasmids/padhe2.gif) | | | |
|  | Promoter: BBa\_J23118 | crRNA: BBa\_K4662022 | tracrRNA: BBa\_K4662031 | Terminator: BBa\_K4662010 |
|  | Backbone: pJUMP29-1D(sfGFP) | | | |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4662/wiki/plasmids/ppfla1.gif) | | | |
|  | Promoter: BBa\_J23119 | crRNA: BBa\_K4662011 | tracrRNA: BBa\_K4662031 | Terminator: BBa\_K4662008 |
|  | Backbone: pJUMP29-1B(sfGFP) | | | |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4662/wiki/plasmids/ppflb2.gif) | | | |
|  | Promoter: BBa\_J23100 | crRNA: BBa\_K4662015 | tracrRNA: BBa\_K4662031 | Terminator: BBa\_K4662009 |
|  | Backbone: pJUMP29-1C(sfGFP) | | | |

#### Test

For the testing we furthermore increased the sample volume for the testing, and decreased the water content in the buffer to have an increased sample: buffer ratio.
Since we switched the buffer conditions, we tested the level 1 plasmids as three biological replicates and two technical replicates.

Surprisingly the effect observed in [Figure 14](#Figure14) was less prominent than we had expected.
The standard deviations are very small, which supports that the knocking down of multiple enzymes simultaneously leads to a constant and efficient flux redirection towards lactate.

![](https://static.igem.wiki/teams/4662/wiki/results/average-level-2.png)

Figure 14.
Assembly of PflA1, PflB1, AdhE1 and AdhE2 sgRNAs to knock down multiple genes at the same time.

#### Learn

The combination of the guide RNAs leads to more available pyruvate in the cell.
This allows future users to redirect the flux of e.g.: glycolysis into a desired direction.

To characterize the functional sgRNAs in more detail, a mobility shift assay could be performed.

{% for sgrna\_source in sgrna\_sources %}

[{{ sgrna\_source.names }} *{{ sgrna\_source.title }}*](%7B%7B%20sgrna_source.link%20%7D%7D)

{% endfor %}

---

## Conclusion / Take-aways

We managed to assemble and test all the components necessary for the function of our final product.

This includes:

* Constructing a QS system in E. coli
* Conditionally expressing proteins in response to C4-HSL
* Increasing Lactic acid production by expression of a recombinant Lactate dehydrogenase
* Increasing lactic acid production through metabolic flux redirection with sgRNAs and CRISPRi
* Optimizing the expression of sgRNA through two rounds of engineering cycle.

Due to failed chromosomal integration, we were unable to test all of the components of our system in one bacterium.

However, chromosomal integration in E. coli is performed on a routine basis, and is likely to succeed in a repeat experiment.

{% endblock %}

{% set sources = [
{"names": "[1.] O'Hanlon DE, Come RA, Moench TR.", "title": "Vaginal pH measured in vivo: lactobacilli determine pH and lactic acid concentration (2019).", "link": "https://doi.org/10.1186/s12866-019-1388-8"},
{"names": "[2.] Tannock GW, Fuller R , Smith ASL.", "title": "Plasmid profiling of members of the family Enterobacteriaceae, lactobacilli, and bifidobacteria to study the transmission of bacteria from mother to infant (1990).", "link": "https://journals.asm.org/doi/abs/10.1128/jcm.28.6.1225-1228.1990"},
{"names": "[3.] Ravel J, et al.", "title": "Vaginal microbiome of reproductive-age women (2010).", "link": "https://doi.org/10.1073/pnas.1002611107"},
{"names": "[4.] Juares Tomas MS, Nader-Macias ME.", "title": "Effect of a medium simulating vaginal fluid on the growth and expression of beneficial characteristics of potentially probiotic lactobacilli.", "link": "https://www.researchgate.net/profile/Maria-Nader-Macias/publication/237506519\_Effect\_of\_a\_medium\_simulating\_vaginal\_fluid\_on\_the\_growth\_and\_expression\_of\_beneficial\_characteristics\_of\_potentially\_probiotic\_lactobacilli/links/0c960537a4815260a1000000/Effect-of-a-medium-simulating-vaginal-fluid-on-the-growth-and-expression-of-beneficial-characteristics-of-potentially-probiotic-lactobacilli.pdf"},
{"names": "[5.] Tietz K, Klein S.", "title": "Simulated Genital Tract Fluids and Their Applicability in Drug Release/Dissolution Testing of Vaginal Dosage Forms (2018).", "link": "https://dissolutiontech.com/issues/201808/DT201808\_A04.pdf"},
] %}
{% extends "layout.html" %}
{% block title %} Experiments {% endblock %}
{% block icon %} ![Experiments Icon](https://static.igem.wiki/teams/4662/wiki/icons/coat.png) {% endblock %}
{% block lead %} **Our Lab Work: Research and Protocols** {% endblock %}
{% block page\_content %}

The following contains the experimental highlights of our project.
The Protocols linked [here](https://static.igem.wiki/teams/4662/wiki/important/lactoback-protocols.pdf) are only improved version, which lead to an optimization of the experiment or the project plan.

We want to point out that this is a condensed version of our lab journal for simplicity and reproducibility for future iGEM teams.
If any questions arise or additional information is needed, we can provide a more extensive list of experiments that we can share if required.

---

## Bacterial Growth Curves

The core of our idea revolves around the apparent advantage that *Lactobacilli* exhibit in acidic surroundings and the preferred environment of our helper bacterium (*E.coli* at a higher pH) [[1.]](#References).

To ascertain the bacteriological growth patterns at a pH 4.5 and pH 6.5 we had to:

* Find a medium where all bacteria species grow at low and high pH
* Finding agar plates were each species show colonies which can be counted, ideally they are also selective for each strain
* Establish a protocol to perform colony forming unit (CFU) measurements to model the growth curves

To demonstrate the growth condition in natural surroundings:

* Experiment with different vaginal fluid simulant media
* Preparing overnight culture at pH 4.5 and pH 6.5 with aerobic and anaerobic conditions to screen for the best growth conditions of the bacteria

continue reading

At the very heart of our concept is the selective advantage *Lactobacilli* seem to have in acidic environments; while lowering the vaginal pH through production of lactic acid, our *Lactobacilli* maintain an environment in which it can thrive.
While several studies have already proven the fact that some *Lactobacillus* strains do better at a lower pH, we wanted to test whether this is also true for the strains we used [[1.]](#References).

We therefore wanted to compare the growth of *Lactobacillus* at different pH levels to the growth of *Escheria coli* and *Bifidobacteria*, as both can also be present in the vaginal microbiome [[2.]](#References).
As microbial composition can vary greatly based on different factors, ethnicity included, we performed the experiments with two different *Lactobacilli* species: *Lactobacillus crispatus* and *Lactobacillus gasseri*, both were obtained via a probiotic pill sponsored by Yoni solutions.
The bacteria present in the probiotic were obtained from a healthy vagina.
Thus the behavior of the bacteria is comparable to the bacteria present in the native genital tract.
While *Lactobacillus crispatus* is the most prevalent species in caucasian women, and makes up a big part of the microbiome in asian, black and hispanic women as well, *Lactobacillus gasseri* too is widespread throughout all ethnicities.
By including both species in our experiments, we wanted to make sure our solution would be applicable to a wide range of patients and not be restricted to one ethnicity only.

We expected all bacteria to be able to grow at both pH values, however, the *Lactobacilli* strains should out-compete the other candidates at low pH environments [[3.]](#References).

First we had to settle on a media in which all four species grow at low and high pH.
After not succeeding with the LB medium we switched to LAPTg, which is preferred by *Lactobacilli*.
All bacteria except Bifidobacteria grew, which is why we decided to try the experiment with *E. coli* and *Lactobacillus crispatus*.

To establish growth curves, we decided to perform colony forming unit (CFU) measurements.
For this purpose, we plated a dilution series of a bacterial culture at several time points.
We decided to use LB-agar plates for *E.coli* and LAPTg-agar plates for the *Lactobacilli*, because they showed optimal growth for colony counting.

At first, we wanted to perform our growth curves in a vaginal fluid simulant.
Unfortunately, none of our tested vaginal fluid simulant media [[4.][5.]](#References) were suitable to perform growth curves, measuring *E. coli* and *L. crispatus* each at high and low pH.
We compromised and decided to measure the growth in VSF, where only the high pH (6.5) condition allowed growth for both bacteria.

---

## Plasmid assembly and measurements

Given the involvement of numerous self-designed plasmids in our concept, successful assembly and transformation of these plasmids was a prerequisite before we could proceed to characterize components and perform measurements.

* Optimizing protocol for golden gate assembly
* Metabolomics analysis using flow injection analysis - mass spectrometry (FIA-MS) to confirm the expression RhlI
* Testing the efficiency of sgRNA by measuring the D-lactic acid concentration in the supernatant
* Characterisation of the LDH with a bioluminescent assay detecting L-lactic acid
* Determining the optimal concentration of C4-HSL for inducing expression via the LasB promoter

---

For more background information on our assembly strategies visit the [Engineering Page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D).

{% for source in sources %}

[{{ source.names }} *{{ source.title }}*](%7B%7B%20source.link%20%7D%7D)

{% endfor %}

{% endblock %}

{% extends "layout.html" %}
{% block title %} Results {% endblock %}
{% block icon %} ![Results Icon](https://static.igem.wiki/teams/4662/wiki/icons/achieved.png) {% endblock %}
{% block lead %} {% endblock %}
{% block page\_content %}

## Achievements

**Proof of concept:** Favourable growth environment for E.coli at high pH (pH ~6.5).

**Design using compuational predictions:** Design and testing of nine sgRNA for flux redirection into increased lactate production.

**Modular engineering:** Building a level 2 sgRNA part to knock down of multiple enzymes simultaneously, leading to a constant and efficient flux redirection towards lactate.

**Verification:** Successful production and secretion of C4-HSL and confirmation by FIA-MS analysis.

**Improved documentation:** Further characterisation of the LasB promoter by induction via RhlI and RhlR.

**Implementation:** Integration of a new lactate dehydrogenase enzyme into *E. coli* which produces favourable L-lactate isomers promoting vaginal health.

**(Preparations for) genomic integration:** Design and amplification of a homology arm pair to knock out the ackA gene.

## Scientific basis: Bacteria growth

Although extensive effort was made to reproduce a functional vaginal fluid simulant (VFS), as described in literature, no satisfying results were obtained. Mostly the high pH condition (pH ~6.5) were more likely to show growth of both
*L. crispatus* and *E. coli*, however, the low pH conditions (pH ~4.5) never worked for both bacteria simultaneously.

We performed growth curves measuring liquid culture OD at 600nm, as well as counted CFU for the purpose of plotting growth curves.
**The CFU protocol did not yield a representative growth curve.
In the future, more optimization in this protocol needs to be done, such as adjusting the dilution factor, the media for plating out the bacteria and increasing the measured time intervals** to gain more data points.

We created growth curves in LAPTg medium, a media specifically designed for cultivating *Lactobacillus* bacteria.
**Even at pH at ~6.5, *L. crispatus* outperformed *E. coli***, as shown in Figure 1.
We kept these results for the sake of replicating them, if we were able to successfully finish our project and co-culture both bacteria in the end.
**Obtaining co-cultures was also attempted, but did not lead to any useful results since the bacterial colonies were not distinguishable on the agar plates**.
Improvements for the future would
**include more screenings for selective media or genetic manipulation by inserting fluorescent labels to distinguish bacteria by color**.

![Growth curve E. coli in LAPTg](https://static.igem.wiki/teams/4662/wiki/results/smooth-growth-curve-experiment-63.png)

![Growth curve L. crispatus in LAPTg](https://static.igem.wiki/teams/4662/wiki/results/smooth-growth-curve-experiment-64-od-al.png)

Figure 1: Comparison of the growth behaviour between *E. coli* (left) and L. crispatus (right) in LAPTg.

Eventually we decided on a VFS medium that allows us to illustrate the growth behaviour in dysbiotic conditions, shown in Figure 2.
The **liquid OD 600nm measurements were successful and nicely confirmed one aspect of our concept:
If we were to use *E. coli* as a probiotic target, it will be able to grow better at dysbiotic conditions, such as high pH**.
This allows us to utilise the engineered quorum sensing system and induce conditional lactate production, as shown further down.

![Growth curve L. crispatus, E. coli in VSF media](https://static.igem.wiki/teams/4662/wiki/results/ec-lc-vsf.png)

Figure 2: The lines represent the growth of *E. coli* and *L. crispatus* in the specific condition.
The dark colors represent the high pH condition, the light colors represent the low pH condition.

Both bacteria show **growth at high pH of the medium, where *E. coli* clearly outcompetes *L. crispatus*, supporting the use as a probiotic for dysbiotic conditions.
Despite literature findings, we could not confirm that *L. crispatus* favours low pH conditions**, this will be part of future research to screen for more suitable media representing the vaginal environment.

## sgRNA design and engineering

We **designed nine different guide RNAs to knock down genes of interest that also use pyruvate as a substrate**, and thus **re-directed the flux of glycolysis to increase lactate production**.

As mentioned in the engineering part, our initial single guide RNA design did not work out due to the fact that our PCR fragments were too short to perform the PCR clean up with the spin columns.
We tested the ethanol purification and unfortunately only one out of nine assembly was confirmed by a positive sequencing result.

The rest contained many mutations or either PCR or assembly didn't work out.

Confirmed sequencing of our designed plasmid after PCR and ethanol purification:

![sgRNA PCR verification](https://static.igem.wiki/teams/4662/wiki/results/sgrna-pcr.png)

Therefore, we redesigned, reordered and reassembled our multiple single guide RNAs with great success.

The sgRNAs were expressed constitutively, while the dCAS9 enzyme was expressed on a tetracycline inducible promoter.
For the measurement of D-lactate, we used a kit that allows us to measure the extinction coefficient of NADH at 340 nm over time and thus we could quantify the lactate concentration in our sample.
The standard curve, as shown in Figure 3, has a high R2 value, showing high accuracy of the kit.

![lactate assay calibration line](https://static.igem.wiki/teams/4662/wiki/results/lactate-calibration-line.png)

Figure 3: Standard curve for D-lactate measurement

The lactate production of each transformed bacterium was measured in two conditions: "uninduced" as a baseline how much lactate is produced normally and “induced” by 5 ug/ml tetracycline.
Figure 4a shows nicely that **the expression of each guide RNA leads to an increase in lactate production**.
It is evident that the lactate concentration drops in the 19.5h timestep.
We hypothesise that upon starvation of the bacteria, the nutrients are not enough to support growth anymore, so the bacteria start to catabolize the lactate within the medium.
**There was only a 10% difference between efficiency predicted at the design stage, and real efficiency**.
With our results, we are neither able to support nor deny the same efficiency order as the predictions.

![lactate assay calibration line](https://static.igem.wiki/teams/4662/wiki/results/level1-sgrna-grid.png)

Figure 4a. Subplots A-I show lactate concentration changes after the insertion of a level 1 sgRNA part targeting pflA (A-C),
pflB (D-F) or adhE (G-I) in the engineered *E. coli*, in the induced versus uninduced state.

Finally, we **wanted to maximise the lactate production by creating a level 2 vector, containing 4 different sgRNAs to efficiently target every protein of interest.**

Based on the results obtained from the individual sgRNA, targeting area and predicted efficiency, we decided to assemble pflA1, pflB1, adhE1 and adhE2.

Surprisingly, the effect observed in Figure 4b was less prominent than we had expected.
The standard deviations are very small, which supports the idea that **knocking down multiple enzymes simultaneously leads to a more consistent flux redirection towards lactate**.

![Assembly of pflA1, pflB1, adhE1 and adhE2 sgRNAs to knock down multiple genes at the same time](https://static.igem.wiki/teams/4662/wiki/results/average-level-2.png)

Figure 4b. Assembly of pflA1, pflB1, adhE1 and adhE2 sgRNAs as a level 2 part which knocks down
multiple genes simultaneously. The standard deviations of measurements at every timestep are reduced compared to
those seen in the respective sublots of constituent level 1 parts in Figure 4a.

We conclude that the **sgRNA design was successful and can be used as a reliable tool to manipulate the central metabolism in the future**.
The combination of the guide RNAs leads to more available pyruvate in the cell, which future users can use to redirect the flux of e.g. glycolysis into a different desired direction.

## Quorum Sensing system implementation

Characterising the quorum sensing system was a vital part of our project, as it was our fundamental approach to having an inducible system.

**Since the vaginal environment for the bacteria is highly dynamic and still poorly characterised, we steered away from an inducible system by e.g. pH**.
Quorum sensing is based on population density and thus allows a robust control of the switch-like activation of its downstream transcription.
Originally found in *Pseudomonas aeruginosa*, we implemented the “Rhl quorum sensing” into our engineered bacterium.
The quorum sensing requires two parts, the production and the sensing unit.

### 1. RhlI

RhlI is a protein that produces a small molecule called *N-butanoyl-L-homoserine lactone* (C4-HSL).
This small molecule acts as a signalling molecule and has been described to be well diffusible (1,3).
Using *flow injection analysis mass spectrometry* (FIA-MS), we were able to detect our molecule of interest in the transformed bacteria.
Although we could not quantify the concentration of the molecule, we were able to see a clear difference between the negative control to our engineered *E. coli*.
In Figure 5, the data analysis from the FIA-MS measurement is shown.

![](https://static.igem.wiki/teams/4662/wiki/results/fia-ms-rhli.png)

Figure 5: FIA-MS analysis results at 170 m/z, which corresponds to the deprotonated C4-HSL molecule.
A3 = supernatant of untransformed *E. coli*, B3 = supernatant of engineered *E. coli*, C1 = extracted cell pellet of untransformed bacterium, D1 = extracted cell pellet of engineered *E. coli*

The fact that some signal in the negative control is visible, might be due to the fact that some bigger molecules fragmented during mass spectrometry, or that dimerization of two smaller molecules resulted in a peak at the same molecular mass.
This data shows clearly that the molecule of interest is produced and secreted successfully.

Confirmed sequencing of our designed plasmid:

![](https://static.igem.wiki/teams/4662/wiki/pcr-verification/pcr-rhli.png)

### 2. RhlR and LasB

Now that we confirmed the expression of a functional RhlI enzyme, it is required to be sensed as well.
RhlR is an enzyme that acts as a transcription factor, as soon as the concentration of C4-HSL reaches a certain threshold and can bind to RhlR.
In the bound state, RhlR gets active and induces transcription of specific genes.
Traditionally, the Rhl quorum sensing system uses the RhlAB promoter.
However, this part has already been characterised in iGEM. **We wanted to increase the iGEM parts collection by confirming that RhlR is also able to induce the LasB promoter (2).
This is the consequence of a crosstalk between two different quorum sensing systems**.
This finding is useful for future teams, since this can be **used to finetune the expression of the protein of interest dependent on the quorum sensing** .
However, the strength of the promoters needs to be compared first.

For testing the RhlR ability to bind to the LasB promoter, we used a level 2 plasmid which contains the RhlR and LasB expressing CFP when induced.
We performed a 24h time incubation and measured the fluorescence of CFP over time, to check whether the intensity increases upon adding a higher amount of C4-HSL manually to the medium of the bacteria.
The untransformed bacteria acted as a negative control.

As shown in Figure 6a, the fluorescence already increases 2 hours after induction of the system.
During the exponential phase, the bacteria are metabolically very active and thus the promoter might be leaking some expression of CFP, as we see in the hours 0-16.
After the cells reach saturation, we don't observe a stronger induction of fluorescence at e.g. 1 mg/ml C4-HSL compared to 0.01 mg/ml, since the quorum sensing system is a switch like mechanism, resulting in either complete induction or no induction at all.
The system is very sensitive, becoming completely induced at 0.01 ug/ml C4-HSL.

![Growth curve L. crispatus, E. coli in VSF media](https://static.igem.wiki/teams/4662/wiki/all-results/lasb-cfp-raw.png)

Figure 6a. Raw measurement of fluorescence.
The untransformed *E. coli* also has a fluorescent signal at 429 nm excitation.

![Growth curve L. crispatus, E. coli in VSF media](https://static.igem.wiki/teams/4662/wiki/all-results/lasb-cfp-corr.png)

Figure 6b. Corrected values for the fluorescent signal of the negative control.

Confirmed sequencing of our designed plasmid:

![](https://static.igem.wiki/teams/4662/wiki/pcr-verification/pcr-rhlr.png)

### 3. Autoinduction of the quorum sensing system

The final step for complete autoinduction required a co-transformation to clone level 2 RhlR and LasB with CFP with the Level 1 RhlI plasmid into one bacterium.
With the CFP output, we measured the fluorescent signal over time to confirm that the CFP signal increases as soon as the population density of the bacteria reaches a threshold density,
which seems to be at OD 0.5. The growth rate of the engineered bacterium is slower, as it reaches the log phase around 2h later than its wild-type counterpart.
In Figure 7, we can see that this resulted in complete characterization of our Rhl-Las quorum sensing hybrid system and is easily adapted by exchanging the CFP moiety to the protein of interest.
Despite the slower growth rate and a ~30% lower stationary population density than observed in the wild-type,
the engineered bacteria outputs nearly double of the fluorescence of the wild-type bacterium.

![CFP output of wild-type vs. induced engineered <i>E. coli</i> MG1655](https://static.igem.wiki/teams/4662/wiki/results/combined-od-cfp-24h-corrected.png)

Figure 7. CFP output of wild-type vs. induced engineered *E. coli* MG1655 (left), as well as simultaneously measured OD values of the two populations (right).
At the 6 hour mark, the fluorescence of engineered *E. coli* overtakes that of the uninduced one
due to the production of CFP at a threshold density of OD ~0.5.

Confirmed sequencing of our designed plasmid:

![](https://static.igem.wiki/teams/4662/wiki/pcr-verification/pcr-cfp.png)

## Lactate dehydrogenase

To increase lactate production in *E. coli*, we **inserted an additional lactate dehydrogenase enzyme, which originates from *Streptococcus bovis* and is a L-lactate dehydrogenase**.
There were several reason why we chose this enzyme:
**first, it has already been successfully overexpressed in *E. coli*;
second, it produces L-lactate not naturally present in high quantities in *E. coli*;
third, the L-lactate may be more favourable for the vaginal microbiome** (4).
To test the efficiency of our L-LDH, we used a L-lactate kit that gave luminescence readouts of luciferin, which was measured and used to quantify the amount of L-lactate in the sample.

As a negative control, we were not only able to compare with untransformed bacteria, but also an LDH knockout strain obtained from the KEIO knockout library.
We used this strain to express our LDH under a constitutive promoter.
Additionally, **we characterised the lactate production dependency on the LasB promoter**, which can be seen in Figure 8.
Surprisingly, the constitutive promoter did not yield high amounts of lactate.

As a next step, we would have exchanged the promoter for a stronger unit and repeated the experiment.
As expected, the induction of the LasB promoter yielded good results, as this had already been characterised with the CFP expression beforehand.

![Growth curve L. crispatus, E. coli in VSF media](https://static.igem.wiki/teams/4662/wiki/all-results/l-lactate-induction.png)

Figure 8. Measurement of L-lactate abundance in control strains; untransformed and knock out. Comparison to induced L-LDH via LasB promoter.

Confirmed sequencing of our designed plasmid:

![](https://static.igem.wiki/teams/4662/wiki/pcr-verification/pcr-lactate-lasb.png)

## Homology arms for chromosomal integration

For the maximal flux redirection, we were planning on **knocking two enzymes out through chromosomal integration**.
We successfully performed PCR to amplify the one homology arms pair out of the bacterial genome.

Confirmed sequencing of our designed plasmid:

![](https://static.igem.wiki/teams/4662/wiki/pcr-verification/pcr-homology-arms.png)

For chromosomal integration, we would have needed the pJUMP47ts plasmid, which we were unable to transform.
Combined with time constraints, we were not able to complete this step.

## Conclusion

*We managed to...*

...show that our vector for a probiotic will have optimal growth conditions in a medium that is designed to stimulate the vaginal environment.

...characterise the RhlR quorum sensing system, while extending the parts library for iGEM.

...computationally model, design and predict resulted a successful set of sgRNAs that efficiently knock down enzymes of the central metabolism and allow redirection of the metabolic flux toward lactate production.

...integrate a new lactate dehydrogenase enzyme into *E. coli* that produces favourable L-lactate isomers to promote vaginal health.

Due to failed pJUMP47ts transformation, we were unable to attempt chromosomal integration and combine the individual parts of the system.

[[1.] Yates et al.  *N-Acylhomoserine Lactones Undergo Lactonolysis in a pH-, Temperature-, and Acyl Chain Length-Dependent Manner during Growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa*  (2002).](https://journals.asm.org/doi/abs/10.1128/iai.70.10.5635-5646.2002)
[[2.] Kumar, L., Patel, S.K.S., Kharga, K., Kumar, R., Kumar, P., Pandohee, J., Kulshresha, S., Harjai, K., Chhibber, S. *Molecular Mechanisms and Applications of N-Acyl Homoserine Lactone-Mediated Quorum Sensing in Bacteria.* Molecules 2022, 27, 7584.](https://doi.org/10.3390/molecules27217584)
[[3.] Tekel S.J., Smith C.L., Lopez B., Mani A., Connot C., Livingstone X., Haynes K.A. *Engineered Orthogonal Quorum Sensing Systems for Synthetic Gene Regulation in Escherichia coli.* Front. Bioeng. Biotechnol., Sec. Synthetic Biology. Volume 7 - 2019.](https://doi.org/10.3389/fbioe.2019.00080)
[[4.] Kraut, J.A.; Kurtz, I. IN-DEPTH REVIEWS *Toxic Alcohol Ingestions, Clinical Features, Diagnosis, and Management. Clinical Journal of the American Society of Nephrology. Volume 3 - 2008.*](https://journals.lww.com/cjasn/Fulltext/2008/01000/Toxic_Alcohol_Ingestions__Clinical_Features)

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