iGEM 2022 — Team go-paris-saclay

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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# ![](https://static.igem.wiki/teams/4370/wiki/small-logo-white.png) Engineering

Contents:

## • Synthetic autotrophy

### Design

We sought to introduce the Calvin Cycle into strains of *Streptomyces*, a genus of bacteria which produces antibiotics along with other highly valuable molecules and specialized metabolites used in laboratories. To introduce the Calvin Cycle into these bacteria, we sought to transfer the genes encoding RuBisCO (Ribulose-1,5-bisphosphate Carboxylase Oxygenase) and PRK (Phosphoribulokinase), as they are the only two missing enzymes that are needed to recreate a variant of the Calvin cycle in heterotrophic organisms (Antonovsky *et al., Cell*, 2016 ; Gassler *et al., Nature Biotechnology*, 2020).

However, to meet this challenge, we had to overcome a major problem: the *Streptomyces* genome has extremely high GC content (72%), which makes the chemical synthesis of genes difficult or impossible. Thus, we had to find a natural source of GC-rich genes encoding RuBisCO and PRK !

First, we thought of using type II RuBisCO (CbbM) from *Rhodospirillum rubrum* ATCC 11170, phosphoribulokinase (prkA) from *Synechococcus elongatus* PCC 7942 as it was previously done in *E. coli* (Antonovsky *et al., Cell*, 2016 ; Gassler *et al., Nature Biotechnology*, 2020). However, due to high GC content in *Streptomyces* (that makes these sequences difficult/impossible to synthesize), we decided to look for versions of those two genes that are already GC rich in nature.

Looking for such a natural source of GC rich genes, we scoured through the genomes of different representatives of the *Streptomyces* genus. As they were previously known to be heterotrophic, we did not expect to find any of our genes of interest. However, we made a discovery : a rather poorly described (draft genome) *Streptomyces* species, *S. bottropensis*, already possesses those two genes! The genes encoding PRK and RuBisCO are located close together and form part of a genomic island surrounded by mobile elements (transposases / recombinases) (**Figure 1**). This fact suggests that the region might have been acquired via a horizontal transfer from a different species.

![](https://static.igem.wiki/teams/4370/wiki/wiki/poc/image6.png)

**Figure 1:** Genomic island of *S. bottropensis*  ATCC 25435 encoding notably RuBisCO and PRK genes

Thus, we thought of using it as a source of genes for our project! The region is about 69% GC rich, thus presenting a close resemblance to genes already present in *Streptomyces*. Furthermore, often GC rich genes cannot be chemically synthesized, so using these already existing genes seemed to us like the most optimal solution.

We also considered using *Carbonactinospora thermoautotrophica* (or *Streptomyces thermoautotrophicus*) since we also discovered that this strain is autotrophic. However, due to the fact that *C. thermoautotrophica* is thermophilic and that according to some sources (Volpiano *et al., Systematic and Applied Microbiology*, 2021), and that it is no longer considered to be a part of the genus of *Streptomyces*, we have decided to use *S. bottropensis* as the source of the genes encoding RuBisCO and PRK in our experiments.

After this brainstorming stage, we developed a roadmap for our cloning strategies described in the document entitled ["IGEM\_CO2\_CURE\_Cloning\_Roadmap"](https://static.igem.wiki/teams/4370/wiki/wiki/footer/igem-co2-cure-cloning-roadmap.pdf).

All the protocols we used to characterize the Streptomyces phenotypes are detailed in the [STREPTObook](https://2022.igem.wiki/go-paris-saclay/education).

### Test

Before implementing the genes from *S. bottropensis* into other bacteria, we decided to do a simple test to see if the species was perhaps naturally autotrophic. If it were autotrophic, we would be sure of the functionality of RuBisCO and PRK from this species. To test it, we simply grew *S.bottropensis* and *S.ambofaciens* (a different species of *Streptomyces* that does not contain PRK or RuBisCO containing genes) in a minimal medium without any carbon source.

The results were quite outstanding since we have observed some growth for both *S. bottropensis* (containing RuBisCO and PRK genes) but also other *Streptomyces* including *S. ambofaciens* (**Figure 2**).

![](https://static.igem.wiki/teams/4370/wiki/wiki/poc/image8.jpg)
![](https://static.igem.wiki/teams/4370/wiki/wiki/poc/image7.jpg)

**Figure 2:** *S. bottropensis* and *S. ambofaciens* grown during 4 days on minimal medium devoid of any carbon source at 30°C under standard atmosphere

However, after restriking those colonies on new plates, we did not achieve the same results: the strains did not grow anymore.

### Learn

This result suggests that *Streptomyces* can initiate a growth in absence of carbon source (maybe thanks to internal carbon stocks) but cannot maintain a prolonged autotrophic growth. Thus we discovered that *S. bottropensis* is in fact not autotrophic, but we decided to continue our testing.

### Test

We have also compared the growth of biomass between those same two strains of *Streptomyces* in standard (0.0415 % CO2) and CO2 enriched (3 % CO2) atmospheres. The final biomass reached by *S. bottropensis* in sub-minimal liquid medium under 3 % CO2 enriched atmosphere was twice the final biomass reached in this medium in a standard atmosphere (**Figure 3**)!

Such a doubling in the biomass under enriched atmosphere was not observed with *S. ambofaciens* (our control strain) grown in the same conditions.

![](https://static.igem.wiki/teams/4370/wiki/wiki/poc/image10.png)

**Figure 3:** Biomass reached after 4 days of culture of *Streptomyces* under standard (0.0415 % CO2) or CO2 enriched (3 % CO2) atmosphere. The media were inoculated with an initial biomass of approximatively 0.04 g.

### Learn

Perhaps high concentration of CO2 is a condition for induction of RuBisCO and PRK coding island expression in *S. bottropensis*. This result further encouraged us to use *S. bottropensis*, a source of our genes of interest.

### Design

We have decided to work on two axes. In the first axis, we attempted to introduce the genes encoding RuBisCO and PRK from *S. bottropensis* into other *Streptomyces* species. In the second axis, we planned to introduce these same two genes optimized for *E. coli* into *E. coli*, in order to further test the functionality of the enzymes.

### Build

All genes of interest were cloned under standard-GC (50 %) or high GC-version (70%) and codon-optimized for an expression in *E. coli* and *Streptomyces*, respectively

We have successfully cloned:

* *cbbL-cbbS* (RuBisCO) ([BBa\_K4370002](http://parts.igem.org/Part%3ABBa_K4370002), [BBa\_K4370003](http://parts.igem.org/Part%3ABBa_K4370003)) optimized for an expression *E. coli* under the control of the *tetO* promoter and an RBS cloned into pGEM-T-easy plasmid.
* *cbbL-cbbS-cbbX* (RuBisCO), operon of *S. bottropensis* ([BBa\_K4370002](http://parts.igem.org/Part%3ABBa_K4370002), [BBa\_K4370003](http://parts.igem.org/Part%3ABBa_K4370003)), wild type under the control of kasOp promoter ([BBa\_K4370006](http://parts.igem.org/Part%3ABBa_K4370006)) and a strong RBS ([BBa\_K4370007](http://parts.igem.org/Part%3ABBa_K4370007)) cloned into pSET152 integrative vector.
* *prk* gene of *S. bottropensis* ([BBa\_K4370005](http://parts.igem.org/Part%3ABBa_K4370005)) under the control of kasOp promoter and a strong RBS cloned in pSET152 integrative vector.
* *prk* optimized for an expression in *E. coli* ([BBa\_K4370005](http://parts.igem.org/Part%3ABBa_K4370005)) under the control of arabinose promoter cloned into pBAD33 plasmid.

### Test

We performed a High Performance Liquid Chromatography (HPLC) on cell extracts of *S. bottropensis* as well as *E. coli* containing a plasmid with genes encoding the small and large subunits of *S.bottropensis* RuBisCO codon-optimized for *E. coli* and a control *E. coli* containing the same vector backbone with a different insert.
To measure RuBisCO activity, RuBP (ribulose-1,5-bisphosphate) - a substrate of RuBisCO was added to the samples, to detect the appearance of RuBisCO’s product - PGA (3-phosphoglycerate). To do it, we measured the absorbance of the samples at 220 nm one minute after the addition of RuBP.
Due to a very high baseline in *E. coli* samples, the results are inconclusive (**Figure 4**). In *S.bottropensis*, we detected a small spike attributed to the appearance of PGA in one of the conditions (**Figure 5**), correlated to the disappearance of RuBP (what is expected for a RuBisCO activity). This result is promising but needs to be confirmed by mass spectrometry analysis (to confirm the identity of the peak attributed to PGA production).

![](https://static.igem.wiki/teams/4370/wiki/wiki/poc/image9.png)

**Figure 4:** HPLC analysis of RuBisCO assay perform with RuBP (ribulose-1-5-bisphosphate) and cell extract of *E. coli* harboring a plasmid encoding *ccbL* ([BBa\_K4370002](http://parts.igem.org/Part%3ABBa_K4370002)) and *cbbS* ([BBa\_K4370003](http://parts.igem.org/Part%3ABBa_K4370003)) extracts under the control of *tetO* promoter ([BBa\_R0040](http://parts.igem.org/Part%3ABBa_R0040)), or control.

![](https://static.igem.wiki/teams/4370/wiki/wiki/poc/image2.png)

**Figure 5:** HPLC analysis of RuBisCO assay perform with RuBP (ribulose-1-5-bisphosphate) and cell extract of *S. bottropensis* grown 4 days at 30°C under different conditions.

### Learn

We have only performed this procedure once, due to time constraints so we suspect a problem with the execution of the experiment. The experiment would have to be repeated to gain more substantial information about the functionality of RuBisCO from *S. bottropensis*. However, the experiments conducted previously give us a lot of hope about the future development of the project.

### Test

To test the activity of the PRK enzyme from *S.bottropensis*, we analyzed the impact of its overexpression in *E. coli* chassis. Indeed, Parikh et al. (Parikh *et al., Protein Eng Des Sel,* 2006) previously reported that the overexpression of the PRK from *Synechococcus* PCC7942 is toxic in this chassis when arabinose is used as a carbon source.

A codon-optimized version of the prk gene of *S. bottropensis* ATCC 25435 ([BBa\_K4370004](http://parts.igem.org/Part%3ABBa_K4370004), with an RBS in [BBa\_K4370005](http://parts.igem.org/Part%3ABBa_K4370005)) was cloned under the control of an RBS ([BBa\_K4370005](http://parts.igem.org/Part%3ABBa_K4370005)) into the L-arabinose-inducible expression vector pBAD33. This plasmid or an empty pBAD33 vector (‘pBAD33-Ø‘) were introduced in two *E. coli* K12 genetic backgrounds, one being able to use arabinose as a carbon source (DH5α), the other not (BW25113). Bacteria were grown on M9 minimal (Sambrook and Russell, 2001) agar plates (supplemented with 1 mM MgCl2, 0.1 mM CaCl2 and 0.1 % thiamine) including 0.2 % glucose and/or arabinose and 30 µg/ml chloramphenicol.

After two days of growth, we observed that the induction of the expression of [BBa\_K4370005](http://parts.igem.org/Part%3ABBa_K4370005) is toxic to *E. coli*, only when the cells use arabinose as the only carbon source. The effect is actually only visible at the lowest cell densities (maybe because at higher density, arabinose concentration is lower).

![](https://static.igem.wiki/teams/4370/wiki/wiki/engineer/image9.png)

**Figure 6**: Growth of *E. col*i strains harboring pBAD33-BBa\_K4370005 or a control vector on M9 supplemented minimal medium in the presence of glucose and/or arabinose. Five µl of bacteria resuspended in M9 medium devoid of carbon source were spotted on the plate. The first dilution (‘10-1’) was adjusted to OD600 nm 0.4 for all strains. The experiments were performed independently for ara+ (DH5) and ara- (BW25113) strains.

### Learn

This confirms that the PRK from *S. bottropensis* is active, and impacts *E. coli* metabolism when arabinose is the only source of carbon as the PRK from *Synechococcus* PCC7942 does.

In the course of this work, we designed a prk sequence optimized for an expression in *E. coli* (BBa\_K4370004, with an RBS in BBa\_K4370005). After constructing and testing the vectors, we learned that the expression of the biobrick is slightly toxic to these cells when arabinose is used as a source of carbon, as previously reported for another PRK. We learnt from this experiment that the genomic island present in *S. bottropensis* ATCC 25435 encodes a functional PRK enzyme. Thus it gives us hope that this key gene of the Calvin cycle could be introduced into other *Streptomyces* in order to implement carbon fixation.

Overall, we think that the experiments done to characterize both RuBisCO and PRK from *S. bottropensis* give a positive outlook on the feasibility of the project and that with more time and resources, our ultimate goal of producing antibiotics from CO2, could be achieved.

![](https://static.igem.wiki/teams/4370/wiki/wiki/engineer/image10.png)

**Figure 7:** Resume of the engineering approach of the implementation of synthetic autotrophy

## • Alternative Cloning Module

### Design

*Streptomyces* are chassis of great biotechnological interest to produce antibiotics but also antitumors, anti-cancer drugs, immunosuppressive agents, etc. Their genome is linear and highly rich in GC (circa 72 %). Their genetic modification generally relies on the introduction of plasmids by conjugation from *E. coli* to *Streptomyces*. It is therefore necessary to develop specific vectors (conjugative, GC rich, specific promoters) in order to use *Streptomyces* as chassis. Because of the challenging nature of working with *Streptomyces*, we have decided to clone the gene encoding PRK in two integrative vectors, pSET152 (mentioned earlier) and pOSV805.

In 2019, Aubry and collaborators published a set of 12 standardized and modular vectors (pOSV801 to pOSV812) designed to integrate genes of interest at specific loci in *Streptomyces* genomes (**Table 1**).

![](https://static.igem.wiki/teams/4370/wiki/wiki/engineer/image1.png)

**Table 1:** List of the *Streptomyces* vectors of the pOSV collection

These vectors are composed of 5 modules: i) the replication module, ii) the antibiotic resistance cassette module (apramycin, hygromycin or kanamycin resistance), iii) the origin of transfer module, iv) the integration module (into PhiBT1, PhiC31, pSAM2 or VWB integration sites), and v) the cloning module. In this collection, this latter does not contain any *Streptomyces* promoter.

We designed a part containing the *Streptomyces* constitutive strong promoter KasOP\* ([BBa\_K4370006](http://parts.igem.org/Part%3ABBa_K4370006)) followed by a RBS from of capsid protein obtained from *Streptomyces* temperate phage ϕC31 ([BBa\_K4370007](http://parts.igem.org/Part%3ABBa_K4370007)) that has been previously described as highly efficient to promote translation when combined with the kasOP\* promoter (Bai *et al., PNAS*, 2015). The part also contains a cassette for the expression of mRFP1 ([BBa\_K4370008](http://parts.igem.org/Part%3ABBa_K4370008)) under the control of an *E. coli* promoter ([BBa\_K1155000](http://parts.igem.org/Part%3ABBa_K1155000)) and an RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)). This RFP expression module is followed by two stop codons and two terminators (BBa\_B0010 and BBa\_B0012). This cassette is meant to be replaced by the construction of interest and offers an easy means of visual screening the clones (pink if the *E. coli* cassette is still present in the plasmid i.e. if the clone contains the parental plasmid, white if not)(**Figure 1**).

![](https://static.igem.wiki/teams/4370/wiki/wiki/engineer/image5.png)

**Figure 8**: Genetic organization of pOSV805 vector and of its modified version harboring [BBa\_K4370010](http://parts.igem.org/Part%3ABBa_K4370010) part

Since [BBa\_K4370010](http://parts.igem.org/Part%3ABBa_K4370010) contains two promoters (one active in *Streptomyces*, one active in *E. coli*), we had to check first that there was no promoter interference and that the mRFP1 gene could be properly expressed from this module at a level sufficient to allow a visual screening for further applications.

### Build

We first introduced our biobrick (kasOP\_RBS\_rfp) into the pGEM-T-easy plasmid via TA cloning and cloned it in *E. coli*. Then, we digested this plasmid containing our biobrick, as well as the integrative plasmid - pOSV805, with the *Not*I restriction enzyme. This allowed introducing the biobrick into the integrative vector at the cloning module site (**Figure 8**).

### Test

We observed the color of the colonies on the plates. We could clearly distinguish red and white colonies, harboring or not the [BBa\_K4370010](http://parts.igem.org/Part%3ABBa_K4370010) part, respectively (**Figure 9**). Thus, whereas bacteria pOV805 were blue, the bacteria transformed by pOSV805- BBa\_K4370010 were now red.

![](https://static.igem.wiki/teams/4370/wiki/wiki/engineer/image8.png)

**Figure 9**: *E. coli* colony phenotype after transformation with a ligation mix harboring pOSV805-BBa\_K4370010 or pOSV805 devoid of cloning module

### Learn

From this experiment, we learnt that pOSV805-BBa\_K4370010 can now be used as a chassis vector for further cloning of gene of interest for a constitutive expression in *Streptomyces*, the red/white screening being a convenient means of screening the clones containing the new construction. Additionally, we learnt that the presence of the promoter active in *Streptomyces* does not interfere with the expression of the cassette optimized for *E.coli*.

#### References

Antonovsky N, Gleizer S, Noor E, Zohar Y, Herz E, Barenholz U, Zelcbuch L, Amram S, Wides A, Tepper N, Davidi D, Bar-On Y, Bareia T, Wernick DG, Shani I, Malitsky S, Jona G, Bar-Even A, Milo R. "Sugar Synthesis from CO2 in *Escherichia coli*." Cell. 2016 Jun 30;166(1):115-25. <https://doi.org/10.1016/j.cell.2016.05.064>
Monal R. Parikh, Dina N. Greene, Kristen K. Woods, and Ichiro Matsumura, “Directed evolution of RuBisCO hypermorphs through genetic selection in engineered *E.coli*”, Protein Engineering, Design and Selection 2006 Mar; 19(3): 113–119 <https://doi-org.insb.bib.cnrs.fr/10.1093/protein/gzj010>
Céline Aubry, Jean-Luc Pernodet , Sylvie Lautru, « Modular and Integrative Vectors for Synthetic Biology Applications in *Streptomyces* spp”, Appl Environ Microbiol, 2019 Aug 1;85(16):e00485-19 <https://doi.org/10.1128/AEM.00485-19>
Chaoxian Bai , Yang Zhang , Xuejin Zhao, Yiling Hu, Sihai Xiang, Jin Miao, Chunbo Lou, Lixin Zhang “Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in *Streptomyces*”, Proc Natl Acad Sci U S A , 2015 Sep 29;112(39):12181-6. <https://www.pnas.org/doi/10.1073/pnas.1511027112>

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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# ![](https://static.igem.wiki/teams/4370/wiki/small-logo-white.png) Experiments

# Protocols

We developed a roadmap for our cloning strategies described in the document entitled ["IGEM\_CO2\_CURE\_Cloning\_Roadmap"](https://static.igem.wiki/teams/4370/wiki/wiki/footer/igem-co2-cure-cloning-roadmap.pdf). All the protocols we used to characterize the *Streptomyces* phenotypes and genome contents are detailed in the [STREPTObook](https://2022.igem.wiki/go-paris-saclay/education). The results related to the characterization of the chassis of the CO2CURE project is in the the [Results](https://2022.igem.wiki/go-paris-saclay/results) webpage. The results concerning the biobricks of the project are presented in detail in the [Engineering page](https://2022.igem.wiki/go-paris-saclay/engineering) as well as on the Registry which is accessible according to a biobrick by biobrick presentation from the [Part page](https://2022.igem.wiki/go-paris-saclay/parts). The complete STREPTObook with all protocols, strain comparison and bioinformatics analyses can be downloaded here:

[LINK to the STREPTObook PDF](https://static.igem.wiki/teams/4370/wiki/wiki/streptobook/igem-co2-cure-streptobook.pdf)

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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# ![](https://static.igem.wiki/teams/4370/wiki/small-logo-white.png) Results

Contents:

## Introduction

On this page of the WIKI, we'll present to you the results related to the characterization of the chassis of the CO2CURE project: *Streptomyces*. The results concerning the biobricks of the project are presented in detail in the [Engineering page](https://2022.igem.wiki/go-paris-saclay/engineering) as well as on the Registry which is accessible according to a biobrick by biobrick presentation from the [Part page](https://2022.igem.wiki/go-paris-saclay/parts).

## Chassis Registry

We conducted experiments to perform bioinformatics and phenotypic analyses (growth, antibacterial bioassays) as described in the [STREPTObook](https://2022.igem.wiki/go-paris-saclay/education) on seven strains of *Streptomyces*. The main results are summarized in the following table.

![](https://static.igem.wiki/teams/4370/wiki/wiki/results/image4.png)

**Legend of the table**: Except for [b] and [c], the color code is as follows: the bluer, the larger; the redder, the smaller. Results from [e], [f] and [g] correspond to the mean values of two independent experiments.

aSMBGCs (specialized metabolite biosynthetic gene clusters) were predicted using [AntiSMASH 6.0](https://antismash.secondarymetabolites.org/#!/start).

bGeneration time was determined in exponential phase after inoculating 106 spores in 10 ml of MP5 medium at 30°C under a regular atmosphere. Color code: the bluer, the shorter; the redder, the longer

cLag phase in MP5 (as described in b) as considered as short if < 12h (blue), or long if > to 12 h (red).

dThe biomass (wet weight) was measured after 5 days of growth as described in [b].

eAntibacterial efficiency (ABE) (defined as the size in cm of the halo of inhibition of *Micrococcus luteus* growth) was determined after 3 days of growth as described in [b]. Antibiotic production (defined as the size in cm of the halo of inhibition of *M. luteus* growth) was determined after inoculating 5.106 spores on MP5 solid medium, and growth during 3 days at 30°C under a regular atmosphere.

fABE was determined after inoculating 5.106 spores on sub minimal medium devoid of carbon source solid medium, and growth during 3 days at 30°C under a regular atmosphere.

gABE was determined after inoculating 5.106 spores on minimal medium containing 0.5 % glycerol (an industrial waste) devoid of carbon source solid medium, and growth during 3 days at 30°C under a regular atmosphere.

hThe conjugation was performed for all strains in conditions optimized for *Streptomyces ambofaciens* engineering (page 11 of the [STREPTObook](https://static.igem.wiki/teams/4370/wiki/wiki/streptobook/igem-co2-cure-streptobook.pdf)).

Most of these experiments were performed only once due to time constraints. Moreover, errors could have crept into the bioinformatics analysis despite our efforts to be vigilant. These results are therefore transmitted with caution and deserve to be confirmed by the reproduction of the results and analyses by future members of the iGEMers community!

## Strain Comparison

Based on all these results, our team wished to understand which of our strains is the best suited to carry the role of the main chassis in the **CO2CURE** project.

**• GC percent**
All strains have a high proportion of GC in their genome (average 72.2 %). Therefore, it will not be a distinguishing criterion on which we will base our opinion. We will have to keep in mind that it means that to perform PCRs, the temperature of melting is high due to the high stability of GC bonds. Moreover, genes inserted in these genomes will require to be GC rich.

**• Genome size**
The *Streptomyces* genome is large: for instance, an *E. coli* K12’s genome is only 4.6 Mb long, almost only half the size of the one from *Streptomyces rimosus* ATCC 10970’s one. *Streptomyces albidoflavus* J1074 has the smallest genome, which can make it a chassis of choice in the context of approaches that aim to make a minimal *Streptomyces* genome. However there is no clear correlation between the genome size and the generation time or the number of SMBGCs. The size of the genome is therefore not an essential criterion for our project.

**• SMBGCs** (Specialized Metabolite Biosynthetic Gene Clusters)
At first sight, we might think that the more SMBGC the strain has, the more interesting the strain is. However, it’s not that simple. On one hand, if you prefer to maximize the production of diversified molecules, the most interesting strain is the one with the higher number of SMBGCs. This is *S. rimosus* ATCC 10970 with 42 SMBGCs, followed by *Streptomyces venezuelae* ATCC 10712 with 31. In this case, you should look for lab conditions that favor SMBGC expression (not always easy to identify…). In this context, the strains may be interesting as a source of new specialized metabolites.

On the other hand, we are often only interested in producing a type of antibiotic of interest whose pathway is either already present in the strain or can be introduced by genetic engineering. In this case, the best chassis can be the one with interesting natural capacities (e.g. production of spiramycin by *S. ambofaciens*, a strain used in the industry for this purpose).

Alternatively, the best chassis for heterologous expression of an SMBGC could be the one with the lowest SMBGCs. Indeed, this has at least two advantages: 1) it limits the consumption of metabolite intermediates for the production of specialized compounds other than the product of interest, 2) it avoids purifying too many contaminating specialized metabolites with the product of interest. In this case, *S. albidoflavus* J1074 could be considered as the best chassis.

**• Genome potential regarding autotrophy**
In order to add autotrophy in these organisms, we want to modify our strains by introducing the GC-rich sequences encoding RuBisCO and PRK. Fortunately, the genome of a *Streptomyces*, namely *S. bottropensis* ATCC 25435, already encodes these genes. Therefore, this strain can be both a good chassis and a source of genes for our project.

**• Growth**
We then decided to find out the generation rate and lag phase of each strain. The faster our strain grows, the faster we can pursue our work. On average, the generation time of all our strains is around 3 hours. The fastest strains were *S. rimosus* ATCC 10970 and *S. bottropensis* ATCC 25435 with a growth rate equal to 2.3 hours. The slowest was *Streptomyces lividans* TK24 with 4.4 hours. However, during our experiment, we discovered another parameter to determine the fastest strain, the duration of the lag phase. Indeed, the lag phase can be short (less than 12h) and long (more than 12h). This new criterion allowed us to separate the strains according to the duration of their lag phase. Taking into account both the generation time and the lag phase, the best chassis is *S. rimosus* ATCC 10970. Finally we evaluated the final biomass of the strains. *S. bottropensis* ATCC 25435 and *S. venezuelae* ATCC 10712 reach the highest biomass levels. However, more than the biomass, it is the production of antibiotics that interests us above all, which we discussed in the following section.

**• Antibiotic production and impact of CO2**
To find the best chassis for antibiotic production we carried out several experiments to compare different aspects of seven *Streptomyces* species, the results of which are summarized in our [STREPTObook](https://2022.igem.wiki/go-paris-saclay/education). All of the strains naturally produce different antibiotics and other secondary metabolites, but we wanted to see which one would be the best candidate to introduce the Calvin Cycle into.

In one of our experiments, we grew all of the seven strains on different media, some of which contained additional CO2 and then carried out a bioassay, in which we poured an indicator species - *Micrococcus luteus* around the *Streptomyces* colony, to observe how *Streptomyces* inhibits the growth of *M. luteus*. The results are usually in a form of a halo, the size of which indicates the efficiency of the biostatic effect of the antibiotics produced by *Streptomyces* (**Figure 1**, **Figure 2**).

We used three media: i) MP5 rich medium (optimized for the antibiotic production by *S. ambofaciens* ATCC 23877 – This medium contains 36 % glycerol, and therefore can be used to recycle industrial waste), ii) a minimal glycerol medium and iii) a subminimal medium without carbon source (as part of the objective of the project).

![](https://static.igem.wiki/teams/4370/wiki/wiki/poc/image1.png)

**Figure 1:** Results obtained by two Bioassays for antibacterial activity of different *Streptomyces* strains on sub-minimal medium (devoid of carbon source), on minimal medium + 0.5% glycerol, or on a rich medium optimized for antibiotic production by *S. ambofaciens* (MP5 medium containing 36 % glycerol) under regular atmosphere (0.0415 % CO2) or under 3 % CO2 (‘+CO2’ in the graph). The error bars correspond to the SEM (standard error to the mean) of two independent experiments.

![](https://static.igem.wiki/teams/4370/wiki/wiki/poc/new-figure-7.png)

**Figure 2:** Antibacterial activity of some *Streptomyces* on plates with MP5 under regular atmosphere. Scale: the diameter of a plate is 8.5 cm.

From the results (**Figure 1**, **Figure 2**), we can see how the antibiotic production differs from strain to strain and medium to medium. We report for the first time the impact of CO2 on antibiotic production by *Streptomyces*. For most strains, the antibacterial activity tends to decrease in the presence of 3% CO2.This decrease remains modest in most cases, so this provides proof of concept that antibiotic production in a CO2-enriched atmosphere is possible.

Overall, from the results we can see that *S. rimosus* and *S. venezuelae* are generally more efficient in antibiotic production than other strains.

## CO2 Fixation

One of the first experiments we carried out was growing different *Streptomyces* species on a minimal medium containing no carbon source. The results were quite outstanding since we have observed some growth for both *S. bottropensis* (containing RuBisCO and PRK genes) but also other *Streptomyces* including *S. ambofaciens* (**Figure 3**).

![](https://static.igem.wiki/teams/4370/wiki/wiki/poc/image4.jpg)
![](https://static.igem.wiki/teams/4370/wiki/wiki/poc/image3.jpg)

**Figure 3:** *S. bottropensis* and *S. ambofaciens* grown during 4 days on minimal medium devoid of any carbon source at 30°C under standard atmosphere

However, after restriking those colonies on new plates, we did not achieve the same results: the strains did not grow anymore. This result suggests that *Streptomyces* can initiate a growth in absence of carbon source (maybe thanks to internal carbon stocks) but cannot maintain a prolonged autotrophic growth. We did not get discouraged!

Beyond that, we have also compared the growth of biomass between those same two strains of *Streptomyces* in standard (0.0415 % CO2) and CO2 enriched (3 % CO2) atmospheres. The final biomass reached by *S. bottropensis* in sub-minimal liquid medium under 3 % CO2 enriched atmosphere was twice the final biomass reached in this medium in a standard atmosphere (**Figure 4**)!

Such a doubling in the biomass under enriched atmosphere was not observed with *S. ambofaciens* (our control strain) grown in the same conditions. A big step for the project. Perhaps this is a condition for induction of RuBisCO and PRK coding island expression in *S. bottropensis*.

![](https://static.igem.wiki/teams/4370/wiki/wiki/poc/image10.png)

**Figure 4:** Biomass reached after 4 days of culture of *Streptomyces* under standard (0.0415 % CO2) or CO2 enriched (3 % CO2) atmosphere. The media were inoculated with an initial biomass of approximatively 0.04 g.

## Genetic engineering

We performed conjugation assays to test the ability of our strains to be modified following a standard protocol. We performed a conjugation from *E. coli* to *Streptomyces* of a test plasmid (pOSV805, Aubry et al., Appl Environ Microbiol, 2019) containing a gene for antibiotic resistance, using a protocol that was previously optimized for *S. ambofaciens* (**Figure 5**). All of the strains, except for *S. rimosus*, had successfully obtained the plasmid. These results indicate that all strains except *S. rimosus* can be easily genetically modified with an integrative plasmid and therefore are suitable for use as chassis. Despite promising results regarding antibiotic production in low carbon conditions, *S. rimosus* is not an optimal chassis regarding the conjugation assay. This means that this strain should either be eliminated from our list of potential candidates for the CO2CURE project, or that further experiments should be conducted to optimize the conjugation protocol for this strain. Fortunately, we were able to identify a publication in which an optimized protocol for this species had been published (Song *et al., J Zhejiang Univ Sci B.*, 2019)). The *S. rimosus chassis* therefore remains potentially interesting.

![](https://static.igem.wiki/teams/4370/wiki/wiki/poc/image5.jpg)

**Figure 5:** Example of conjugation of pOSV805 integrative plasmid into *S. venezuelae*. The colonies correspond to exconjugants that have acquired the resistance marker (hygromycin resistance).

## What makes a good chassis? survey

We then wondered what would other iGEM team pick as the best chassis among our strains. To answer such a question, we checked at the results from a form we transmitted to all iGEM teams. In this form, we asked all iGEM teams to rate from 1 to 5 some criteria to determine which characteristics from strains would be the most important to characterize to complete a chassis registry. While this book is being written, eleven iGEM teams have answered this form (**Figure 6**).

![](https://static.igem.wiki/teams/4370/wiki/wiki/collaborations/image4.png)

**Figure 6:** Results of the “What makes a good chassis?” survey, the teams that answered are Lund, Wageningen, Groningen, Duesseldorf, SUNY Oneonta, Pui Ching Middle School (2 times), NTHU\_Taiwan, iGEM Aachen, IISc Bengaluru, iGEM IONIS.

While team teams rarely gave a rating of five, they often rated one or two the criteria, so we still can understand which ones are the most important. Among the most important ones, there is the generation time, final biomass of the production, the sensitivity of the chassis to the possible toxicity of the synthetic system and natural metabolic potential/properties of the chassis. Considering the generation time and the natural potential/properties of the chassis, *S. rimosus* ATCC 10970 is the best strain. Considering the final biomass, the best one would be *S. bottropensis* ATCC 25435/DSM40262.

## Final Conclusion

Having all of this in mind, we think that *S. rimosus* ATCC 10970 is the best strain to conduct the CO2CURE project. It’s extremely high antibiotic production in all media accompanied by its short generation time and lag phase makes it the strain we must privilege. *S. bottropensis* ATCC 25435 is also quite interesting to consider since it encodes RuBisCO and PRK. This strain could be interesting as a chassis for SMBGC heterologous expression.

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