iGEM 2022 — Team dtu-denmark

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In the scientific community it is important that results can be replicated. In order to achieve this our lab books and protocols are shown below as PDF. So even you can replicate our work.

* Lab books

## Menu

[Introduction](#Header1)
[Gene expression in *A. niger*](#Header2)
[Furfural converting enzymes](#Header3)
[Fungal Promoters](#Header4)
[Fungal Synthetic Transcription Factors](#Header5)
[Collaborations](#Header6)
[Modeling notebook](#Header7)

## Introduction

Find the relevant lab book, and download it by clicking the link.

## Gene expression in *A. niger*

[Lab book 1](https://static.igem.wiki/teams/4129/wiki/notebook/ges-assembly-of-crispr-plasmids-for-nig1-and-nig3-integration.pdf) - Cas9 and donor plasmid assembly
[Lab book 2](https://static.igem.wiki/teams/4129/wiki/notebook/ges-transient-expression-plasmid.pdf) - asembly of transient expression plasmid

## Furfural converting enzymes

[Lab book 1](https://static.igem.wiki/teams/4129/wiki/notebook/fec-a-niger-transformation-with-arz7774-fuco-and-hmfh.pdf) -*A. niger* transformation with plasmids containing furfural converting enzymes
[Lab book 2](https://static.igem.wiki/teams/4129/wiki/notebook/fec-assembly-of-arz7774-fuco-and-hmfh-for-chromosomal-integration-in-a-niger.pdf) - Assembly of plasmids with furfural containing enzymes
[Lab book 3](https://static.igem.wiki/teams/4129/wiki/notebook/fec-testing-furfural-degrading-enzymes-in-a-niger-growth.pdf) - Cultivation of *A. niger* mutants
[Lab book 4](https://static.igem.wiki/teams/4129/wiki/notebook/fec-testing-furfural-degrading-enzymes-in-a-niger-hplc.pdf) - HPLC analysis
[Lab book 5](https://static.igem.wiki/teams/4129/wiki/notebook/fec-testing-the-germination-capabilities-of-strains-biolector.pdf) - Testing the germination of *A. niger* mutant spores

## Fungal Promoters

[Lab book 1](https://static.igem.wiki/teams/4129/wiki/notebook/funpro-new-plasmids-plan-for-native-promoters-testing.pdf) - New fungal promoter testing
[Lab book 2](https://static.igem.wiki/teams/4129/wiki/notebook/funpro-promoter-insertion-strategy.pdf) - Promoter insertion strategy

## Fungal Synthetic Transcription Factors

[Lab book 1](https://static.igem.wiki/teams/4129/wiki/notebook/synthetic-transcription-factors-stf-a-niger-integration.pdf) - *A. niger* integration
[Lab book 2](https://static.igem.wiki/teams/4129/wiki/notebook/synthetic-transcription-factors-stf-assembly.pdf) - FunsTF assembly
[Lab book 3](https://static.igem.wiki/teams/4129/wiki/notebook/synthetic-transcription-factors-stf-characterization.pdf) - FunsTF characterization
[Lab book 4](https://static.igem.wiki/teams/4129/wiki/notebook/synthetic-transcription-factors-stf-mutagenesis.pdf) - FunsTF mutagenesis

## Collaborations

[Lab book 1](https://static.igem.wiki/teams/4129/wiki/notebook/collabs-interlab-study-2022.pdf) - Interlabs study 2022
[Lab book 2](https://static.igem.wiki/teams/4129/wiki/notebook/collabs-synthetic-transcription-factors-stf-tu-delft.pdf) - Synthetic transcription factor - TU Delft

## Modeling notebook

Dry lab modeling notebook [Dry lab book](https://2022.igem.wiki/dtu-denmark/model) - Modeling

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On this page we describe the engineering design cycle of a number of subprojects: Gene expression in *Aspergillus niger*, Furfural converting enzymes, Fungal promoters and Synthetic transcription factors
The engineering design cycle consists of four phases: Design, Build, Test and Learn, or DBTL for short.

* Gene Expression in *Aspergillus niger*
* Furfural Converting Enzymes
* Putative inducible fungal promoters
* Synthetic Transcription Factors

## Menu

[Introduction](#Header1)
[Cycle 1](#Header2)
[Cycle 2](#Header3)
[Cycle 3](#Header4)
[References](#Header5)

## Introduction

In order to test our synthetic transcription factors (sTFs) and furfural converting enzymes (FCE), we had to engineer *Aspergillus niger* to express them. There are several methods to do this, with varying levels of complexity and time required. The easiest and quickest method is to use a transient expression platform (TEP) while a more consistent and stable solution is genomic integration. Initially, we attempted genomic integration of an expression cassette, as we wanted to characterise our proteins in more stable clones. A TEP could potentially be developed later.

## Cycle 1

### Design

We based our expression cassette and method of genomic integration on methods described in the book chapter: “Genomic Editing: CRISPR-Cas9” by Hoof et al. 2018. Two different types of plasmids were designed; a plasmid expressing cas9 and gRNA, as well as a donor plasmid.

#### *Cas9- and gRNA-expressing plasmid*

This plasmid has a cas9 gene and a robust gRNA expression system, see figure 1, which is compatible with multiplexing. It is structured such that gRNAs are flanked by tRNAs, enabling the release of gRNAs from the transcript by polymerase III (Nødvig et al. 2018). A hygromycin resistance gene enables selection after transformation, increasing the efficiency of the system and an AMA1 element (BBa\_K3046024) is also included, enabling efficient replication in *A. niger*. Lastly, elements such as origin of replication and ampicillin resistance encoding genes are present for *E. coli* work (BBa\_K3385073).

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/engineering-success/gene-expression-figure1.png)
 *Figure 1: Cas9 plasmid, with inserted protospacer for IS-nig1 (BBa\_K4129030)*

#### *Donor plasmid*

The expression cassette has the promoter PgpdA (BBa\_K4129024) and terminator TtrpC (BBa\_K678036) and is USER compatible, thus allowing insertion of a gene of interest (GOI) in the expression cassette, see figure 2 for plasmid with inserted *arz7774* (BBa\_K4129007). Downstream of the expression cassette is the selection marker *pyrG* (BBa\_K3385072), which can be used for selection in *pyrG* defficient *A. niger* during cultivation on minimal media. *pyrG* facilitates uridine and uracil synthesis pathways. The selection marker is flanked by double-repeat regions, facilitating the excision of the selection marker by counter-selection. Double repeat regions are unstable parts of the genome and can be spontaneously excised unless selected for. This event can also be enhanced by counter selection towards the gene flanked by the double repeats. This makes it possible to use the same selection marker for several rounds of transformations. Gene expression cassette and pyrG with double repeats are flanked by homologous regions of two kilobases each, each flanking the target-site in *A. niger*, allowing for precise and efficient integration into the fungal genome. The plasmid shares the same *E. coli* elements as the Cas9 plasmid.

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/engineering-success/gene-expression-figure2.png)
 *Figure 2: Donor plasmid map, with inserted arz7774 (BBa\_K4129018)*

### Build

Both of the above plasmids were developed in-house. Multiple Cas9 plasmids were assembled via USER cloning, each with a different protospacer for the gRNA. Multiple plasmids were assembled to increase the chance that at least one plasmid had an efficient gRNA. A donor plasmid for each FCE was also assembled via USER cloning.

### Test

In order to save time, we did not assess the efficiency of each gRNA, but went ahead with cotransformation of Cas9 and donor plasmid. Several colonies were obtained, which were streaked clean on new plates. Tissue PCR was performed, but we could not validate the entire expression cassette was inserted, even after several attempts with changes made to the method and extra controls.

### Learn

Validating the genomic insertion of the expression cassette was much harder than expected. Several changes to the system could be made to skip this step. For instance, if the expression cassette had a selection marker, this could be screened for.
Alternatively we could turn to the less stable solution and develop the TEP. This requires no tissue PCR to validate the expression system, as PCR can be performed before the transformation.

## Cycle 2

### Design

Due to time constraints, we decided to develop the TEP as the next step, see figure 3 for plasmid map. Our TEP has several components that enable easy construction in *E. coli*, while also being somewhat stable in *A. niger*, granted it is selected for. A USER cassette flanked by the promoter PgpdA and terminator TtrpC enables easy construction using USER cloning. An AMA1 element enables *A. niger* to replicate the plasmid and the pyrG selection marker was also present to allow selection of correctly transformed mutants. An ori element and ampR enables stability in E. coli.

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/engineering-success/gene-expression-figure3.png)
 *Figure 3: TEP plasmid map, with inserted Arz7774 (BBa\_K4129027)*

### Build

The elements used to construct our TEP were amplified by PCR with USER-primers. The expression cassette was amplified from the previously described donor plasmid (figure 2) and the plasmid backbone from another in-house plasmid. Amplicons were analysed on gels and assembled to our TEP using USER cloning.

With our final TEP, we could now insert our GOIs into the expression cassette via USER cloning.

### Test

The testing was two-fold as we had to test our sTFs as well as our FCE. All *A. niger* clones survived on minimal media meaning they were successfully transformed and had the plasmid conveying uridine and uracil synthesis pathways present.

Clones with FCE were characterised using a BioLector for cultivation and sTF clones were cultivated in 96 well plates and on agar plates.
The results were of mixed success; we did not prove the FCE worked, but some sTFs did show activity. Thus the TEP worked for our initial search of usable sTFs.

### Learn

While we knew a TES is not as stable as a genomic insertion, it was a simple way to express proteins. With the data we have gathered through the use of TESs, we could conclude that it was a beneficial platform to develop, as we did get some useful data out of it.
However, we still needed a more robust expression platform, to further characterise our proteins.

## Cycle 3

### Design

While the first Cas9-based design of a genomically integratable expression cassette could not be proven to be inserted, other Cas9-based genome integration systems exist. The DIVERSIFY platform (Jarczynska et al. (2021)), developed in-house at DTU, can be used to create mutants easily validated by screening due to the presence of a marker in the expression cassette. This is useful as this circumvents the bad performance of our tissue PCR.

The DIVERSIFY system requires more time, as two transformations are needed before the desired clones have been made. This led to us to initially go with a simpler Cas9-based system, as described in the first design phase.

The DIVERSIFY system consists of two plasmids, as well as two Cas9 plasmids.

#### Common synthetic gene integration site (COSI) plasmid

This plasmid is used to establish the expression cassette in the genome. It has a similar structure to the donor plasmid described in the first design phase, but with additional elements, see figure 4. The same promoter and terminator encoding genes are used, but a screening gene β-glucuronidase (*uidA*) from *E. coli* is also inserted. This expression cassette is flanked by small homology regions facilitating gene expression cassette exchange. This segment is then flanked by the larger homology regions which were also described in the first design phase, facilitating genomic insertion into *A. niger*. Downstream of the gene expression cassette, between the small and large homology region, the selective gene *pyrG* is present.

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/engineering-success/gene-expression-figure4.png)
 *Figure 4: COSI plasmid map.*

This plasmid contains the expression cassette with the GOI. There are two versions of this plasmid; the one we would use for FCE includes promoter and terminator, while the second one does not. Small homology regions facilitate cassette exchange with the integrated platform.

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/engineering-success/gene-expression-figure5.png)
 *Figure 5: GEC plasmid map with empty expression cassette.*

#### Cas9 plasmids

The only difference between the two Cas9 plasmids needed to use the DIVERSIFY system is the gRNA used. One plasmid is used to integrate the DIVERSIFY platform into the genome, while the other is used for cassette exchange, by targeting the *uidA* gene.

With these plasmids, we would be able to integrate the DIVERSIFY platform and use uidA to screen for colored colonies, while later inserting our GOI with the GEC plasmid, now looking for non-colored colonies, as the uidA would be deleted (figure 6).

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/engineering-success/gene-expression-figure6.png)
 *Figure 6: Overview of the DIVERSIFY platform. A) shows the expression cassette exchange by uidA targeting by Cas9 and GEC as the donor. Small homology regions facilitate this exchange, shown as A1 and B1. B) shows the screening process used to find clones with exchanged expression cassettes. For the integration of the DIVERSIFY platform, the screening plate would be the opposite, so control (C) would not be colored, and surrounding colonies would be. Figure 1 from Jarczynska et al. (2021).*

### Build

Due to time constraints, we did not get to assemble our DIVERSIFY plasmids.

## References

Hoof, J.B., Nødvig, C.S., & Mortensen, U.H.. Genome Editing: CRISPR-Cas9. In: de Vries, R., Tsang, A., Grigoriev, I. (eds) *Fungal Genomics. Methods in Molecular Biology*, vol 1775, (2018)

Nødvig, C.S., Hoof, J.B., Kogle. M.E., Jarczynska, Z.D., Lehmbeck, J., Klitgaard, D.K., & Mortensen, U.H.. Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli. *Fungal Genetics and Biology*, 115, 78-89. (2018)

Jarczynska, Z.D., Rendsvig, J.K.H., Pagels, N., Viana, V.R., Nødvig, C.S., Kirchner, F.H., Strucko, T., Nielsen, M.L. Mortensen, U.H.. DIVERSIFY: A Fungal Multispecies Gene Expression Platform, *American Chemical Society*, 10, 579-588. (2021)

## Menu

[Introduction](#Header6)
[Cycle 1](#Header7)
[References](#Header8)

## Introduction

The objective of this subproject was to identify, assemble and express FCE in *A. niger*, and thus lower the sensitivity to furfural.

## Cycle 1

### Design

Looking through literature, several candidate genes for conversion of furfural into other compounds were identified. Specifically, two conversion reactions were identified, in which either furfuryl alcohol or furoic acid were produced from furfural. As literature showed furoic acid might be less toxic than furfuryl alcohol, we chose two putative enzymes that could convert furfural to furoic acid (*arz7774* and *hmfH*), and one that could convert furfural to furfuryl alcohol (*fucO*). The enzymes have been registered as: *arz7774* (BBa\_K4129007), *fucO* (BBa\_K4129008) and *hmfH* (BBa\_K4129009)

*arz7774* is a putative aldehyde dehydrogenase from *Amorphotheca resinae*, another filamentous fungi and was identified by Wang. et al. (2015), *fucO* is an Lactaldehyde oxidoreductase from *Escherichia coli* identified by Koopman. et al. (2013) and *hmfH* is a putative oxidoreductase from *Cupriavidus basilensis* identified by Zheng et al. (2010)

### Build

The amino acid sequence of the enzymes were identified and a DNA sequence was generated and codon optimised for *A. niger* by using GenScripts GenSmart Codon Optimization tool. These were ordered as gene blocks from IDT.

Two rounds of cloning were performed, as we first tried to use the genomically inserted expression cassette, as described in the section: Gene expression in *Aspergillus niger*.
As the insertion could not be validated by tissue-PCR, we switched to the TEP. Both cloning methods involved USER cloning, thus the lab work was near identical.

With USER cloning, the plasmid with *fucO* could not be constructed, thus only two FECs were built with *arz7774* and *hmfH*.

### Test

Several rounds of testing were conducted using a BioLector and HPLC samples were analysed. There was no indication that our *A. niger* clones with FEC had a higher tolerance towards furfural compared to wildtype *A. niger*.

Subsequent HPLC analysis of the growth media as well as samples containing a predefined amount of furfural showed no traces of furfural, meaning that the equipment possibly had been calibrated wrongly prior to testing.

### Learn

As we could not prove the FECs had a beneficial effect on *A. niger*, it begs the questions, why? The chosen FECs are putative and might not fold correctly in *A. niger*. The enzymes could also be transported to an incorrect compartment in *A. niger*, as the current build does not include a localization-peptide.
It could also be beneficial to test the enzyme in vitro, if we can replicate the required conditions and have the necessary substrates available.

Adding a localization-peptide would be quite easy to do, using the current TEP. However, it is hard to say if that would lead to the enzymes behaving as we initially expected.

## References

Koopman, F., Wierckx, N., Winde, J.h., & Ruijssenaars, H.J. Identification and characterization of the furfural and 5-(hydroxymethyl)furfural degradation pathways of *Cupriavidus basilensis* HMF14. *Proceedings of the National Academy of Sciences*. 107(11), 4919-4924 (2010)

Zheng, H., Wang, W., Yomano, L.P., Geddes, R.D.,,Shanmugam, K.T.& Ingram, L.O. Improving Escherichia coli FucO for Furfural Tolerance by Saturation Mutagenesis of Individual Amino Acid Positions. *Applied and Environmental Microbiology*, 79(10), 3202-3208, (2013)

Wang, X., Gao, Q. & Bao, J. Transcriptional analysis of *Amorphotheca resinae* ZN1 on biological degradation of furfural and 5-hydroxymethylfurfural derived from lignocellulose pretreatment. *Biotechnol Biofuels* 8, 136 (2015)

## Menu

[Introduction](#Header9)
[Cycle 1](#Header10)
[Cycle 2](#Header11)

## Introduction

The objective was to evaluate the activity of three fungal promoters (scFDH1, BBa\_K4129001; anFDH, BBa\_K4129002; scFDH2, BBa\_K4129003) in *A. niger* when exposed to furfural.

## Cycle 1

### Design

To test the activity of the fungal promoters in *A. niger*, we decided to transiently express BBa\_K4129004, BBa\_K4129005, BBa\_K4129006 in a plasmid based system. As a plasmid vector for our constructs we chose BBa\_K3046021 as we had access to it in our lab.

### Build

The plasmids were assembled by USER cloning and replicated in *E. coli*. Initial validation of the plasmids by restriction digestion showed clear discrepancies between the assembled plasmids and the maps of the designed ones. However, BBa\_K3046021 had been successfully used at DTU in 2019 and Sanger sequencing showed that all essential components of our constructs (AMA1 plasmid replication region, fungal selection marker, and prokaryotic selection marker) were present. We concluded that the discrepancies between the assembled and designed plasmids were due to minor errors in the maps, and that the assembled plasmids were functionally equivalent to the designed one. Therefore we proceed to test our constructs in *A. niger*.

### Test

Multiple attempts at transforming our plasmids into *A. niger* failed. After confirming that the *A. niger* protoplasts used were viable, we concluded that our plasmids were not functional.

### Learn

After realising that the stock of BBa\_K3046021 we had was not reliable, we began a new iteration of the DBTL cycle taking care of thoroughly validating each component before settling on which design to build.

## Cycle 2

### Design

We redesigned our plasmids using BBa\_K4129006 as the backbone. BBa\_K4129006 was selected as we were able to validate its sequence and successfully transform it into *A. niger*.

### Build

The new plasmids were successfully assembled, validated, and transformed into *A. niger*.

### Test

The *A. niger* strains carrying the plasmid expression systems were grown in liquid media. Subsequently, half of the samples were induced with furfural. Fluorescence and absorbance were monitored to quantify the mCherry reporter protein and biomass produced by the strains. Multiple negative and positive controls were included. Of particular interest for the learning phase of this cycle were the two positive controls which carried BBa\_K3046004 either on a plasmid or as a genomic insertion as the allowed for a comparison of the two expression systems.

### Learn

The results showed that the three fungal promoters tested were neither constitutive nor induced by furfural in *A. niger*, see the results [here](https://2022.igem.wiki/dtu-denmark/results#Header4). As the goal of this track was to investigate the activity of these promoters during furfural exposure, the engineering cycle was not repeated. Out of the positive controls, the strain carrying a genomically integrated BBa\_K3046004 showed higher average fluorescence and lower variance compared to its plasmid expression system counterpart. This indicates that when investigating promoter activity, strains with genomic insertions are preferable over transient expression systems.

## Menu

[Cycle 1](#Header14)
[Cycle 2](#Header15)
[References](#Header16)

## Cycle 1

### Design

The sTFs are a part of the synthetic expression system (SES) we developed. The design of the sTFs will thus depend on the SES. The SES are designed to be a double fungal expression system, where the sTF will be constitutively expressed from the sTF expression unit and regulate the expression of the reporter cassette. The sTF expression unit consists of the fungal promoter PgpdA (BBa\_K4129024), a sTF like FunsTF05 (BBa\_K4129103), and the fungal terminator TcgrA (BBa\_K3669007). The other functional unit of the SES is the rapporter cassette, which consists of the synthetic promoter 6xLexO-gpdA-minimal promoter (6x-LexO-Pmin) (BBa\_K4129115), mCherry (BBa\_J06504) and the fungal terminator TtrpC (BBa\_K678036).

Our synthetic transcription factors were designed with a modular approach. This was to allow easy engineering of the sTFs due to the fact that the sTFs are composed of three different functional domains fused together with 2 linker regions. The three domains are; 1) a DNA-binding domain, 2) a ligand sensing domain and 3) a transactivation domain. The following domains were used for the modularity: One DNA-binding domain, namely LexA, two ligand sensing domains, namely HbaR and Hmox1 and lastly two transactivation domains, namely B112 and VP16. Lastly, the linker between DNA-binding domain and ligand sensing domain can be one of two possible modules: a short version and a longer version. This gives eight possible sTFs in total. Each sTF also includes the nuclear localization sequence (NLS) SV40, which ensures transport into the cell nucleus (Garcia-Bustos et al (1991)).

It has not been experimentally proven that HbaR and Hmox1 interact with furfural. A study on Hmox1 indicated through computational analysis that Hmox1 is able to bind to furfural or vice versa (Santhakumar et. al (2021)). HbaR is a transcription factor that can sense benzoic acid and derivatives thereof (Egland et. al (2000, Castaño-Cerezo (2020))). Therefore, to increase the binding affinity of furfural to the sTF, sixteen rational mutations were designed. These additional sixteen ligand sensing domains of HbaR were named HbaR1-16. This means that, in total, 72 different sTF were designed, eight of them were labelled as original sTF and the remaining 64 as HbaR variants.

A more indept discussion about our synthetic transcription factors and promoter can be seen [here](https://2022.igem.wiki/dtu-denmark/stfdesign).

### Build

The assembly of the double expression system was carried out using USER cloning in *E. coli* DH5a. The assembly was standardised so that each sTF could be assembled in bulk using the same protocol. The assembly consisted of 2 rounds of assembly, with the first round of assembly being of the sTF expression units and the sTF reporter cassette. The next round was fusing the two cassettes together on our TEP.

The sTF expression units and the sTF reporter cassette were assembled independently of each other, so when a sTF expression unit was ready to be assembled into the TEP, there would be no bottlenecks.

### Test

The transient expression plasmids was used to find a [proof of concept](https://2022.igem.wiki/dtu-denmark/proof-of-concept). The expression cassettes were tested both on solid media plates and in liquid culture. The media used was minimal media, minimal media with 2 mM benzoic acid and minimal media with 0.6 g/L furfural.

The solid media plates with the *A. niger* carrying the double expression system on TEP was incubated 4 days at 30°C. The fluorescence was assessed in Gel200 Vilber Smart Imagine Fusion FX and it was shown that FunsTF02, FunsTF04, FunsTF05 and FunsTF70 did fluoresce consecutively with different efficiency. See the results [here](https://2022.igem.wiki/dtu-denmark/results#Header5).

The liquid culture testing was performed in a CellStar 48-well cultivation plate. The optical density and fluorescence was measured in a ClarioStar plate reader. The measurements were taken after induction. Results showed that FunsTF05, FunsTF18 and FunsTF70 also displayed fluorescence in liquid.

### Learn

There were some SES where the sTF showed constitutively expressed mCherry. This was a good proof of concept for our SES, but a vast majority of the tested strains did not work. The sTF needs to be further engineered to be induced in response to furfural or benzoic acid. This engineering requires a good assay and the assays performed so far could be improved, especially in liquid media.

## Cycle 2

### Design

The liquid assay could be improved by transferring from transient expression to genome integration, because the expression will be more stable. This system is the [Gene expression](https://2022.igem.wiki/dtu-denmark/engineering#Header4) in *A. niger*. In addition to stable expression, an improvement could be to measure the dynamics of the promoter. This could be achieved by using a BioLector, allowing further investigation of the expression over time as measurements are taken automatically at regular intervals by the machine.

In short, the SES consists of many components which can be optimised. We would focus on the sTF, because it needs to be engineered more in order to be inducible. The first step would be designing a SES without any sTF to understand whether mCherry expression from 6xLexO-Pmin was leaky or not .
The next steps will be more attempts at characterization, especially to understand the ligand sensing domains. We would fuse a GFP to the sTFs to identify their cellular location.
The cellular location will indicate if the sTFs, that did not work were due to the fact they were not in the nucleus or something else. It would also be interesting to remove the SV40 NLS, to see if the sTF is transported into the cell nucleus without the NLS.

## References

Castaño-Cerezo S, Fournié M, Urban P, Faulon JL, Truan G. Development of a Biosensor for Detection of Benzoic Acid Derivatives in Saccharomyces cerevisiae. *Front Bioeng Biotechnol*. 2020 Jan 7;7:372. doi: 10.3389/fbioe.2019.00372. PMID: 31970152; PMCID: PMC6959289.

Egland PG, Harwood CS. HbaR, a 4-hydroxybenzoate sensor and FNR-CRP superfamily member, regulates anaerobic 4-hydroxybenzoate degradation by Rhodopseudomonas palustris. *J Bacteriol*. 2000 Jan;182(1):100-6. doi: 10.1128/JB.182.1.100-106.2000. PMID: 10613868; PMCID: PMC94245.

Erill I, Escribano M, Campoy S, Barbé J. In silico analysis reveals substantial variability in the gene contents of the gamma proteobacteria LexA-regulon. *Bioinformatics*. 2003 Nov 22;19(17):2225-36. doi: 10.1093/bioinformatics/btg303. PMID: 14630651.

Garcia-Bustos J, Heitman J, Hall MN. Nuclear protein localization. *Biochim Biophys Acta.* 1991 Mar 7;1071(1):83-101. doi: 10.1016/0304-4157(91)90013-m. PMID: 2004116.

Hirai H, Tani T, Kikyo N. Structure and functions of powerful transactivators: VP16, MyoD and FoxA. *Int J Dev Biol.* 2010;54(11-12):1589-96. doi: 10.1387/ijdb.103194hh. PMID: 21404180; PMCID: PMC3419751.

Ottoz DS, Rudolf F, Stelling J. Inducible, tightly regulated and growth condition-independent transcription factor in Saccharomyces cerevisiae. *Nucleic Acids Res.* 2014;42(17):e130. doi: 10.1093/nar/gku616. Epub 2014 Jul 17. PMID: 25034689; PMCID: PMC4176152.

Radman M. SOS repair hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis. *Basic Life Sci.* 1975;5A:355-67. doi: 10.1007/978-1-4684-2895-7\_48. PMID: 1103845.

Santhakumar P, Prathap L, Roy A, Jayaraman S, Jeevitha M. Molecular docking analysis of furfural and isoginkgetin with heme oxygenase I and PPARγ. *Bioinformation.* 2021 Feb 28;17(2):356-362. doi: 10.6026/97320630017356. PMID: 34234396; PMCID: PMC8225605.

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## Menu

[Experiments](#Header1)
[→ sTF-promoter pairs](#Header1-1)
[→ Putative inducible fungal promoters](#Header1-2)
[→ Furfural converting enzymes](#Header1-3)
[→ Partnership with TU Delft](#Header1-4)
[→ Adaptive Laboratory Evolution](#Header1-5)
[→ References](#Header1-6)
[Protocols](#Header2)
[→ PCR](#Header2-1)
[→ Gel Electrophoresis](#Header2-2)
[→ DNA fragment purification](#Header2-3)
[→ USER cloning](#Header2-4)
[→ Competent DH5α *E. coli* transformation](#Header2-5)
[→ *E. coli* colony PCR](#Header2-6)
[→ Plasmid validation by restriction digestion](#Header2-7)
[→ *A. niger* protoplastation](#Header2-8)
[→ *A. niger* transformation](#Header2-9)
[→ Fungal tissue PCR](#Header2-10)
[→ Measuring mCherry fluorescence in *A. niger* (liquid)](#Header2-11)
[→ Measuring mCherry fluorescence in *A. niger* (solid)](#Header2-12)
[→ Testing furfural resistance in germinated wild type cells (BioLector)](#Header2-13)
[→ Testing furfural resistance in mutants (BioLector)](#Header2-14)
[→ qPCR](#Header2-X)
[→ Preparing BioLector samples for HPLC analysis](#Header2-15)
[→ Testing germination times in mutants during furfural exposure (BioLector)](#Header2-16)
[→ Adaptive laboratory evolution](#Header2-17)
[→ SDS-page](#Header2-18)

## Introduction

To engineer an Aspergillus niger strain capable of detecting and converting furfural, it is first necessary to develop a functional furfural biosensor and to identify furfural-converting enzymes that retain their activity when heterologously expressed in *A. niger*. To this end, we divided our experimental strategy in three distinct, yet deeply interconnect, experimental tracks:

The design and testing of synthetic transcription factor (sTF) paired with a minimal promoter that could be used to build furfural-inducible expression systems; the testing of activity in *A. niger* of three fungal promoters which were identified as potentially induced by furfural; the heterologous expression in *A. niger* of both prokaryotic and eukaryotic furfural-converting enzymes, and the subsequent evaluation of their activity *in vivo*.

Alongside the main experimental tracks, we performed a smaller experiment to further our partnership with TU Delft, and an Adaptive Laboratory Evolution (ALE) experiment to evaluate if a furfural-tolerant *A. niger* strain could be obtained without any rational engineering.

#### sTF-promoter pairs

The sTFs were designed to be modular proteins, each containing a DNA-binding domain, a putative furfural sensing domain for allosteric regulation and an activator domain. Initially, six sTFs were designed utilising a combinatorial approach to mix and match the different domains (as described in details here). Subsequently, new sTFs were designed to include the mutations identified as beneficial by the in silico [modelling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D). We also designed a minimal promoter containing LexO binding sites for our sTFs utilising the Tet-ON promoter as a blueprint ([Wanka et al.](https://doi.org/10.1016/j.fgb.2015.11.003)).

Four of the initial sTFs were synthesised de novo, and their DNA sequences used as templates for the PCR amplification of their modular domains. These PCR products were used to assemble the remaining two possible sTFs by USER cloning. Similarly, DNA fragments of the modular domains containing the modelled mutations were obtained by PCRs and assembled by USER fusion to build new sTFs.

To test the functionality of these sTFs, we assembled test-device plasmids for transient expression in *A. niger*. Each test-device plasmid carries a constitutively expressed sTF cassette, the minimal promoter regulating the expression of a mCherry cassette, an AMA sequence for replication in *A. niger*, and an auxotrophic selection marker (PyrG). All the test-devices were assembled by USER cloning, replicated in DH5α *Escherichia coli*, and purified using the GenElute™ Plasmid Maxiprep Kit (Sigma-Aldhrich) according to the manufacturer's instructions. Following validation by restriction digestion and Sanger sequencing, the test-devices were transformed into PyrG deficient *A. niger*. These strains were used to carry out an experiment on solid media to obtain a qualitative measure of the sTFs’ activity and one in liquid media for a quantitative measure. For both experiments, the *A. niger* strains were grown either in minimal media, minimal media with furfural, or minimal media with benzoic acids. Subsequently, their mCherry fluorescence was monitored with an imaging system (solid media) or with a plate reader (liquid media).

#### Putative inducible fungal promoters

A previous transcriptomic study ([Li et al.](https://doi.org/10.1128/AEM.00158-21)), showed that the genes Formate Dehydrogenase 1 and 2 (scFDH1 and scFDH2) are upregulated in response to furfural in Saccharomyces cerevisiae. Therefore, we decided to test if the promoters of these genes are responsive to furfural in *A. niger* as well. Through NCBI’s BLAST we identified the homologous gene to scFDH1 and scFDH2 in *A. niger*, and decided to test its promoter too. We amplified the 1001 bp upstream of the start codon of the three genes by PCR using genomic DNA as a template. The fragments were subsequently used to assemble test-device plasmids containing an AMA sequence, a PyrG auxotrophic selection marker, and an mCherry expression cassette under the regulation of the promoters to be investigated. The test-devices were replicated in DH5α *E. coli*, validated by restriction digestion and Sanger sequencing, and transformed in PyrG deficient *A. niger*. These *A. niger* strains were grown in liquid minimal media, induced with furfural and their fluorescence and absorbance were monitored in a plate reader to characterise the promoters’ activity.
The activity of the anFDH promoter was also investigated by qPCR, as we quantified the FDH transcript from a WT strain and the mCherry transcript from our test-device strain following growth in minimal media or furfural supplemented media. The advantage of using qPCR is twofold: when quantifying FDH transcript, it allows characterization of the anFDH promoter in its native regulatory environment; when quantifying mCherry, it reduces noise from background fluorescence and it is more sensitive to small changes in expression.

#### Furfural converting enzymes

Several organisms exist that are either resistant or capable of converting furfural to combat the toxic effects. After an extended literature search we decided to try out three separate genes: fucO, hmfH and arz\_7774 from the organisms Escherichia coli, Cupriavidus basiliensis and Amorphotheca resinae Zn1 respectively. All three genes convert furfural into a less toxic derivative; for FucO, furfuryl alcohol is formed, while HmfH and Arz\_7774 convert furfural into 2-furoic acid.

We were unable to successfully clone FucO, and as such chose to continue only with Arz\_7774 and HmfF for further testing. Next we tried to integrate the two genes into the genomes of a PyrG deficient *A. niger* strain. All transformation attempts failed, and as such we switched to a transient expression system through a plasmid containing an AMA sequence and a PyrG auxotrophic selection marker (BBa\_K4129025).
Several growth experiments were conducted with the two *A. niger* strains transiently expressing furfural converting enzymes and the wild type *A. niger* strain, where growth rates and germination capabilities under various levels of furfural were tested. Subsequently, a HPLC analysis was done to investigate if the engineered strains were capable of converting furfural to 2-furoic acid.

#### Partnership with TU Delft

We aimed at sending purified FunsTF01-FunsTF06 to the TU Delft team, so that they could investigate the binding affinity of our sTFs to our synthetic promoter in vitro. We assembled vectors expressing our sTF with 6xHisTag into a pET28a backbone, and transformed them into *E. coli* BL21(DE3) cells. The sTF were produced in *E. coli*, extracted, and purified using ThermoFisher NiNTA spin columns. However, a subsequent SDS page showed that the proteins had not been purified. Due to time restraints we were not able to repeat the production and purification of our sTFs and never sent them to the TU Delft team for analysis.

#### Adaptive Laboratory Evolution

To evaluate if a furfural-tolerant *A. niger* strain could be obtained without any rational engineering, we carried out an ALE experiment. WT *A. niger* was grown in liquid media containing 0.6 g/L of furfural. When pellets started to form, the sample was used to inoculate a new media containing furfural. This process was repeated several times while slowly increasing the furfural concentration in the media. We expected that furfural tolerant mutants would evolve and outcompete the previous *A. niger* strains. After 4 months, however, a bacterial contamination was detected. The contaminating organisms successfully outcompeted *A. niger* forcing us to interrupt the experiment.

#### References

* Wanka F. et al. Tet-on, or Tet-off, that is the question: Advanced conditional gene expression in *Aspergillus*. *Fungal Genetics and Biology* 89,72-83 (2016)
* Li B. et al.Improving Acetic Acid and Furfural Resistance of Xylose-Fermenting *Saccharomyces cerevisiae* Strains by Regulating Novel Transcription Factors Revealed via Comparative Transcriptomic Analysis. *Applied and Environmental Microbiology* 87 (2021)

## Protocols

#### PCR

Materials:

* Forward primer (10 µM)
* Reverse primer (10 µM)
* Template DNA (10-25 ng for plasmid DNA, 250 ng for genomic DNA)
* PCR grade water
* PCR tubes

Procedure:

 1. Prepare the PCR reaction as indicated below:

| Component | Volume |
| --- | --- |
| Phusion U Multiplex Mix | 25 μL |
| Forward primer | 5 μL |
| Reverse primer | 5 μL |
| Template DNA | 10-25 ng (plasmid) 250ng (gDNA) |
| PCR grade water | to 50 µL of total volume |

 2. Put the sample(s) in a PCR machine and run the following protocol:

|  |  |  |
| --- | --- | --- |
| Step 1 (1x) | 3 min. | at 98°C |
| Step 2 (30x) | 30 sec. | at 98°C |
|  | 30 sec | at \*°C |
|  | \*\* sec. | at 72°C |
| Step 3 (1x) | 5 min. | at 72°C |
| Hold |  | at 10°C |

*\*note: this temperature depends on your primer pair*

*\*\*note: depends on the length of the amplified piece (around 30 sec./kb)*

#### Gel Electrophoresis

*Note: SYBR Safe and gels containing SYBR Safe should always be handled with gloves*

Materials:

* 1% or 2% agarose in TAE buffer
* SYBR Safe (10 000x)
* Gel tray and comb
* Gel electrophoresis chamber
* TAE buffer
* PCR-amplicon
* Loading dye (6x)
* DNA ladder (100 bp or 1 kbp)
* Gloves

Procedure:

* Set up a gel tray and comb on a levelled surface
* Pour 1% or 2% agarose in TAE buffer (at 60°)
* Add SYBR Safe 10 000x to a final concentration of 1x
* Wait until the gel is set (15-20 min.)
* Move the gel to a gel electrophoresis chamber filled with TAE buffer
* Add loading dye (6x) to the samples to a final concentration of 1x
* Load samples in the gel
* Run the gel at 100V for 20min
* Remove gel from the chamber and place it in the Bio-Rad Imager to take pictures

#### DNA fragment purification

*Note: Gels containing SYBR Safe should always be handled with gloves*

Materials:

* All necessary material for gel electrophoresis
* DNAfragments / PCR products
* Scalpel
* 96% ethanol
* Gloves
* Gel Imager/excision plate and goggles
* Gloves
* Gel DNA extraction kit
* 2 ml Eppendorf tubes
* Zymoclean Gel DNA Recovery Kit

Procedure:

* Run the DNA fragments / PCR products on a 1% agarose gel (see Gel Electrophoresis protocol)
* Place gel on the gel imager, turn on gel imager while wearing the goggles
* Cut the desired band out using a scalpel and place it in a pre-weighted 2 ml Eppendorf tube
* When excising multiple bands, clean the scalpel between bands with 96% ethanol
* Use the Zymoclean Gel DNA Recovery Kit following the manufacturer protocol to purify the DNA

#### USER cloning

Materials:

* Purified USER fragment
* Linearized USER vector
* 10x Cutsmart buffer (NEB)
* USER enzyme (NEB)
* PCR grade water

Procedure:

 1. Prepare the USER reaction as indicated below:

| Component | Volume |
| --- | --- |
| 10x Cutsmart buffer (NEB) | 1 μL |
| USER enzyme (NEB) | 1 μL |
| Linearized USER vector | 1 μL |
| USER fragments | equal amount of each in ng |
| PCR grade water | to 10 µL of total volume |

 2. Put the sample(s) in a PCR machine and run the following protocol:

|  |  |  |
| --- | --- | --- |
| Step 1 (1x) | 30 min. | at 37°C |
| Step 2 (8x) | 3 min. | at \*°C |
| Step 3 (1x) | 5 min. | at 16°C |
|  | 10 min. | at 10°C |
| Hold |  | at 4 °C |

*\*note: this temperature starts at 28 C° and decreases by 1°C with each cycle*

 3. Cool the USER reaction on ice if transforming them into *E. coli* right away or store them at -4°C

#### Competent DH5α *E. coli* transformation

Materials:

* Competent DH5α *E. coli* cells
* USER cloning product / plasmid
* Ice
* Heat block
* Selective media plates
* Spreader
* Gloves
* 96% ethanol
* Bunsen burner

Procedure:

* Thaw the competent cells on ice
* Set the heat block at 42°C
* Aliquot 50 μl of competent cells to sterile 1.5 ml Eppendorf tubes
* Add 10 μl of USER cloning product or 500 ng of plasmid DNA to the tube and mix gently
* Incubate on ice for 15 min
* Transfer the tubes to the heat block for 45-60 sec
* Incubate on ice for 15 min
* Plate the cells on selective media. When plating, work near the Bunsen burner flame to prevent contaminations
* Sterilise the spreader by dipping it in ethanol and passing it in the flame between plating samples

#### *E. coli* colony PCR

Materials:

* Taq DNA Polymerase Master Mix RED (2x)
* Forward primer (10 µM)
* Reverse primer (10 µM)
* Milli-Q water
* PCR grade water
* Agar plate with colonies to be tested

Procedure:

 1. Transfer a colony with a pipette tip to a PCR tube with 30 µl MQ and resuspend the colony

 2. Prepare the PCR reaction as indicated below:

| Component | Volume |
| --- | --- |
| Taq DNA Polymerase Master Mix (2x) | 5 μL |
| Forward primer | 1 μL |
| Reverse primer | 1 μL |
| Resuspended colony | 1 μL |
| PCR grade water | 2 µL |

 3. Put the sample(s) in a PCR machine and run the following protocol:

|  |  |  |
| --- | --- | --- |
| Step 1 (1x) | 3 min. | at 98°C |
| Step 2 (30x) | 30 sec. | at 98°C |
|  | 30 sec. | at \*°C |
|  | \*\* sec. | at 72°C |
| Step 3 (1x) | 5 min. | at 72°C |
| Hold |  | at 10 °C |

*\*note: this temperature depends on your primer pair*

*\*\*note: depends on the length of the amplified piece (around 20 sec./kb)*

#### Plasmid validation by restriction digestion

Materials:

* Plasmid
* Relevant restriction digestion enzyme
* Relevant buffer (10x)
* PCR tubes
* Gel imager

Procedure:

 1. Choose a restriction digestion enzyme that will cut the plasmid in a recognisable pattern

 2. Identify the buffer associated with the restriction enzyme that was chosen

 3. Prepare the restriction digestion reaction as indicated below:

| Component | Volume |
| --- | --- |
| Restriction enzyme | 0.5 μL |
| Buffer (10x) | 1 μL |
| Plasmid DNA | 500 ng |
| Milli-Q water | to 10 µL of total volume |

 4. Incubate the reaction at the temperature indicated by the manufacturer for 1 hour

 5. Run the restriction digestion product on a 1% agarose gel (see [Gel Electrophoresis](#Header2-2))

 6. Take pictures of the gel using the gel imager

 7. Compare the observed and expected restriction pattern

#### *A. niger* protoplastation

Materials:

* Plate with desired *A. niger* strain culture
* Sterile 500 ml shake flasks
* Sterile Mira cloth
* YPD with required supplements (e.g. Uridine for PyrG deficient strains)
* Aspergillus protoplastation buffer (APB)
  1.1 M MgSO4 and 10 mM Na-phosphate buffer, pH adjusted to 5.8 with2 M NaOH* Aspergillus transformation buffer (ATB)
  1.2 M Sorbitol; 50 mM CaCl2·2 H2O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 M HCl to 7.2* Sterile Milli-Q water
* Sterile 50 ml falcon tubes
* Sterile 1.5 ml Eppendorf tubes

Procedure:

* Collect the conidia from a plate by adding 5 mL of sterile liquid YPD with required supplements and firmly rubbing the colonies with a sterile Drigalski spatula. The conidial suspension is withdrawn from the plate and added to the 500 ml shake flask containing 100 mL of YPD
* The culture is incubated for 2 days at a30 C° and 150 rpm
* Harvest the mycelia and germlings by using Miracloth
* Wash the mycelia with APB to remove the liquid media from the mycelia
* Resuspend the mycelium in 10-20 ml APB solution containing 40 mg Glucanex/ml APB
* Homogenize mycelial and enzyme suspension gently to obtain the best possible digestion of the fungal cell wall
* Shake at 30°C and 150 rpm for 2-3 hours
* Filter through Miracloth and collect the flow through in a Falcon tube
* Add APB up to the total volume 40 ml
* Carefully make an overlay with 5 ml of 2 fold diluted Aspergillus transformation buffer (ATB)
* Dilute with sterile Milli-Q water
* Centrifuge at 3000xg (acceleration 9, deceleration 4) for 12 min
* Upon a successful protoplastation, a halo of white protoplast slurry is caught just below the surface. Collect protoplast slurry and transfer it to a new tube
* Add ATB up to the total volume 40 ml/li>* Centrifuge at 3000xg (acceleration 9, deceleration 9) for 12 min. Discard supernatant
  * Resuspend the protoplasts in approximately 1 ml ATB
  * Aliquot 150 μL protoplasts into 1.5 Eppendorf tubes and store at -80°C

#### *A. niger* transformation

Materials:

* *Aspergillus niger* protoplasts
* Plasmids
* Aspergillus protoplastation buffer (APB)
  1.2 M Sorbitol; 50 mM CaCl2·2 H2O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 M HCl to 7.2* PCT
  Final conc: 50 % w/vol PEG 8000; 50 mM CaCl2; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.5. Store PCT at 4°C* Plates of osmotically stabilised selective media
* Sterile 1.5 ml Eppendorf tubes

Procedure:

* Gently mix 500 ng of plasmid DNA and 50 μL protoplast in an Eppendorf tube
* Add 150 μL PCT
* Mix by inversion of the tube
* Incubate 10 min at room temperature
* Add 250 μL ATB and mix
* Plate on osmotic stabilized, selective media

#### Fungal tissue PCR

Materials:

* AQ97 High Fidelity DNA Polymerase Master Mix (2x) OR Taq DNA polymerase Master
* Mix Red (2x)
* Primers
* PCR grade water
* Sterile PCR tubes
* Sterile 10 µl tips and pipette

Procedure:

 1. Prepare the PCR reactions as indicated below:

| Component | Volume |
| --- | --- |
| Polymerase Master Mix (2x) | 12.5 μL |
| Forward primer | 2.5 μL |
| Reverse primer | 2.5 μL |
| PCR grade water | 7.5 μL |

 2. Put the sample(s) in a PCR machine and run the following protocol:

|  |  |  |
| --- | --- | --- |
| Step 1 (1x) | 30 min. | at 98 °C |
| Step 2 (30x) | 30 sec. | at 98°C |
|  | 30 sec. | at \*°C |
|  | \*\* sec. | at 72°C |
| Step 3 (1x) | 5 min. | at 72°C |
| Hold |  | at 10°C |

*\*note: this temperature starts at 28 C° and decreases by 1°C with each cycle*

*\*\*note: depends on the length of the amplified piece (around 30 sec./kb)*

 3. Two reactions per sample should be prepared, and will be run with different quantities of tissue

 4. Using a 10µl pipette tip and compatible pipette set to highest volume, penetrate the mycelium and agar (partially) and transfer pipette tip to the first PCR reaction and wash the pipette tip in the PCR solution

 5. Then move the tip (empty) to the second PCR reaction and wash the pipette tip in the PCR solution:

* Work on ice, after taking the tissue sample. Don't forget to mark the sampled colony and PCR tube, so you can find the colony later
* Avoid sampling spores and too much mycelium. This will inhibit the PCR reaction

#### Measuring mCherry fluorescence in *A. niger* (liquid)

Materials:

* Spore suspension (\(10^9\) spore/ml)
* CELLSTAR 48 well suspension culture plate
* Minimal media (liquid)
* Inducer (furfural, benzoic acid)
* Plate reader (ClarioStar)

Procedure:

* Fill each well with 790 µl of minimal media
* Add 10 µl of spore suspension to each well
* Incubate at 30°, 150 rpm for 20h
* Prepare minimal media with 5x inducer:

+ 1x for furfural is 0.6 g/L
+ 1x for benzoic acid is 2mM

* Add 200 µl of minimal media with the 5x inducer
* Incubate at 30°C, 150 rpm for 45 minutes
* Measure mCherry fluorescence and absorbance of the wells
* Repeat step 7 and 8 three more times (4 in total)
* Incubate at 30 C°, 150rpm, for the desired duration of the experiment (e.g. 120)
* Measure fluorescence and absorbance at regular intervals (e.g. 20h)

#### Measuring mCherry fluorescence in *A. niger* (solid)

Materials:

* Spore suspension (\(10^9\) spore/ml)
* Minimal media (liquid)
* Inducer (furfural, benzoic acid)
* Plastic petri dishes
* Imager system (Vilber Fusion FX)

Procedure:

* Prepare minimal media agar plates with the desired inducer with inducer:

+ for furfural is 0.6 g/L
+ for benzoic acid is 2mM

* Place 4 µl droplets of spore suspensions in three point of each plate
* Incubate at 30° for 3 to 5 days
* Take pictures of the plates with an mCherry fluorescence filter (maintain the same exposure time to ensure the pictures can be directly compared to each others)

#### Testing furfural resistance in germinated wild type cells (BioLector)

*Note: Wear gloves and work in the LAF bench when handling furfural*

Materials:

* BioLector
* FlowerPlate 48 well MTP
* YPD media
* Furfural
* *A. niger* spore suspension
* 1 Shake flask with baffles

Procedure:

* Inoculate 100 mL of YPD in a shake flask with wild type *A. niger*
* Grow cell culture shaking at 30°C
* Allow cells to reach a concentration of \(~5\times10^6\) cells per mL
* Once the desired concentration is reached, prepare the Biolector plate:

+ Add 750µL of cell culture to 750µL YPD in each well, and add the desired furfural concentration to each well

* Run the BioLector at settings:

+ 85% humidity
+ 30°C
+ Shaking at 800 RPM
+ Biomass measure at gain = 3

* Stop run after 48 hours and analyse the results

#### Testing furfural resistance in mutants (BioLector)

Materials:

* BioLector
* FlowerPlate 48 well MTP
* Minimal media
* Furfural
* Wild type *A. niger* spore suspension
* Mutant *A. niger* strains spore suspensions

Procedure:

* Prepare the FlowerPlate 48 well MTP plate with 1.3 mL minimal media, with \(~5\times10^6\) spores per mL in the various wells (except for the blanks)
* Run the BioLector at settings:

+ 85% humidity
+ 30°C
+ Shaking at 800 RPM
+ Biomass measure at gain = 3

* Once the exponential phase has started and been going for at least 3 hours, pause the BioLector and add the desired furfural concentrations you wish to test
* Stop run once stationary phase has been reached

#### qPCR

Materials for cultivations and sample preparations:

* Shake flask with baffles
* Minimal media
* Furfural
* Wild type A. niger spore suspension
* Mutant A. niger spore suspension
* Shaking incubator
* Sterile miracloth filters
* Paper towel
* Liquid nitrogen
* Motor
* Aluminium foil
* RNAse-free 2 mL tubes.

Materials for RNA extraction:

* RNeasy® Mini Kit (50), Cat. #74104, QIAGEN
* Gloves
* RNaseZap™
* NanoDrop™ machine for RNA quantification
* Filter tips and pipettes for RNA

Materials for cDNA synthesis:

* BIO-RAD iScript Select cDNA Synthesis Kit, Cat. #1708896
* Filter tips
* Sterile PCR tubes
* PCR machine

Materials for cDNA synthesis:

* BIO-RAD iScript Select cDNA Synthesis Kit, Cat. #1708896
* Filter tips
* Sterile PCR tubes
* PCR machine

Materials for qPCR:

+ Primers pair (designed on NCBI primer-BLAST tool) for each gene of interest as well as a house hold gene, e.g. actin.
+ Agilent Brilliant III Ultra-Fast SYBR®, Green QPCR Master Mix, Cat. #600882
+ Filter tips
+ qPCR plates and seals
+ qPCR machine

Procedure for cultivations and sample preparations:

Prepare cultivation of samples in each condition in triplicates. Inoculate approximately 4x10^6 spores into each shake flask containing 150 mL minimal media. Grow for approximately three days, or until an appropriate amount of biomass has been achieved for all samples. Add 1 g/L furfural to the samples corresponding to that condition. After furfural has worked for 1.5 hours, filter off the biomass using the miracloth filters. Squeeze the fluid out of the biomass by pressing it with paper towels. Wrap the biomass in aluminium foil and flash freeze it using liquid nitrogen. When the biomass is completely frozen, cool a motor with liquid nitrogen while crushing the biomass into a dusty powder. Take a precooled RNase-free 2 mL tube and collect approximately 0.5 mL biomass powder. Proceed immediately to RNA extraction or store samples at -80C until needed.

Procedure for RNA extraction, cDNA synthesis and qPCR:
Clean your workspace using 70% ethanol and RNaseZap™. Extract the RNA as described in the RNeasy® Mini Kit, use gloves and filter tips at all times . Quantify RNA using NanoDrop and note the concentrations and the A60/A80 values to ensure sufficient quality. Synthesise cDNA using BIO-RAD iScript Select cDNA Synthesis Kit. Before running the real qPCR, test at what primers work best and at which concentrations. Prepare a small amount of cDNA from each sample and make a test qPCR (using Agilent Brilliant III Ultra-Fast SYBR® kit) with primer concentrations of 100 mM, 300 mM and 600 mM. Pick the best primers and concentrations and run all your samples as described in the Agilent Brilliant III Ultra-Fast SYBR® Kit.
The relative expression levels were calculated according to the formula 2^(−ΔΔCT).

#### Preparing BioLector samples for HPLC analysis

Materials:

+ HPLC vials
+ BioLector plate with cells culture you wish to sample
+ 2 Eppendorf tubes for each well to sample
+ Ice
+ Acetonitrile (HPLC grade)

Procedure:

+ Once a BioLector run has concluded, transfer the BioLector plate to ice
+ Transfer all liquid from each well to a Eppendorf tube kept on ice (If you wish to test biomass as well, carefully scrape the sides of each well and transfer the biomass to the eppendorf tube as well)
+ Spin down all samples in a centrifuge at >10.000 RPM at 4°C
+ Transfer the supernatant to a new eppendorf tube
+ Mix 125µL sample with 125µL of HPLC grade acetonitrile
+ Samples are now ready for HPLC analysis
+ An Agilent 1290 Infinity II LC system coupled to an Agilent 6545 QTOF MS (Agilent Technologies, Santa Clara, CA, USA) was used to perform UHPLC-HRMS. The column used was an Agilent Poroshell phenyl-hexyl column (2.1 x 150 mm, 1.9µm). MS detection was performed in positive mode on the QTOF MS equipped with Agilent Dual Jet Stream electrospray ion source. The Agilent MassHunter Qualitative Analysis B.07.00 software tool was used for processing and data analysis of MS data

#### Testing germination times in mutants during furfural exposure (BioLector)

Materials:

+ BioLector
+ FlowerPlate 48 well MTP
+ Minimal media
+ Furfural
+ Wild type *A. niger* spore suspension
+ Mutant *A. niger* strains spore suspension

Procedure:

+ Prepare the FlowerPlate 48 well MTP plate with 1.3 mL minimal media, with \(~5\times10^6\) spores per mL in the various wells (except for the blanks)
+ Add the varying levels of furfural you wish to test germination potential at to the wells
+ Run the BioLector at settings:

- 85% humidity
- 30°C
- Shaking at 800 RPM
- Biomass measure at gain = 3

+ Stop run after 48 hours and analyse the results

#### Adaptive laboratory evolution

Materials:

+ Shake flask with baffles
+ YPD media
+ Furfural
+ Wild type *A. niger* spore suspension
+ Shaking incubator

Procedure:

+ Inoculate 100 mL of YPD with \(~1\times10^6\) *A. niger* spores
+ Grow the cells in the flask until the first pellets start to form
+ Transfer 20 mL cell broth to 80 mL fresh YPD in a new sterilised shake flask with baffles
+ Add furfural to a final concentration of 0.6 g/L and let the cells grow for 5 days
+ Repeat the transfer and growth step, while slowly increasing the furfural concentration in the shake flasks by 0.05 g/L every month

#### SDS-page

*Note: SDS gels NuPage Sample reducing agent (10x) contain denaturing agents and should always be handled with gloves*

Materials:

+ Tris-glycine SDS sample buffer (2x)
+ NuPage Sample reducing agent (10x)
+ Protein sample
+ Tris-Glycine SDS-running buffer (10x)
+ Precast SDS gel
+ Appropriate gel chamber (need to match the pre cast gel specifications)
+ Mark12 protein ladder
+ simplyBlue staining agent
+ Gloves

Procedure:

+ Mix the following in a PCR tube:

- 20 µL Tris-glycine SDS sample buffer (2x)
- 4 µL NuPage Sample reducing agent (10x)
- 16 µL of the sample

+ Incubate for 5 min at 95°C
+ Dilute 10x Tris-Glycine SDS-running buffer to 1x
+ Remove the plastic comb and bottom tape from the precast gel
+ Assemble the gel in the electrophoresis chamber
+ Fill the chamber with buffer until the line on the chamber
+ Load 10 µL Mark12 protein ladder
+ Load the gel with 15 µL of sample for well
+ Run the SDS-PAGE for 35-45 min at 225 V constant
+ Rinse the gel with milli-Q water
+ Crack the plastic case and extract the gel
+ Place the gel in a shallow tray and add simplyBlue until the gel is covered
+ Incubate for 1-3 hours at room temperature with gently shaking
+ Discard the staining agent and wash the gel in milli-Q water for 1 hour with gently shaking
+ Discard water and take a picture of the gel

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
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![](https://static.igem.wiki/teams/4129/wiki/top-images/results.svg)
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On this page we show the results from three subprojects: Furfural converting enzymes, Fungal promoters and synthetic transcription factors.

* Synthetic transcription factors
* Putative inducible fungal promoters
* Furfural converting enzymes

## Menu

[Introduction](#Header1)
[Results](#Header2)
[→ Characterising furfurals effect on the growth of A. niger](#Header2-1)
[→ Characterising the effect of furfural on the growth of A. niger mutants](#Header2-2)
[→ Characterization of furfurals effect on the germination of A. niger spores](#Header2-3)

## Introduction

Two different mutants were obtained, each with a different furfural converting enzyme (FCE). These will be annotated as Arz and hmfH and are transformed with the following plasmids BBa\_K4129018 and BBa\_K4129020, respectively. These were then subsequently tested in a BioLector alongside WT *A. niger*.

## Results

We have not been able to experimentally show the FCE working in *A. niger*. However, we have shown that the expression of the FCEs have a detrimental effect on both growth rate and final biomass gain, suggesting they are a metabolic burden to the fungus under stressful environments. This also suggests that *A. niger* has a better way to handle furfural in the media than our proposed pathways enabled by the FCEs, granted they work in *A. niger*.

### Characterising furfurals effect on the growth of *A. niger*

With WT *A. niger* inoculum, wells with differing concentrations of furfural were inoculated. After just seven hours it was clear that furfural in the media had a detrimental effect on the growth rate of WT *A. niger*. This is greatly illustrated on figure 1. But what was also discovered was that WT *A. niger* does not reach the same biomass gain when growing in the presence of furfural as it does without the presence of furfural. This agrees with the literature, and sets the basis for reducing the effect of furfural on fungal growth by giving WT *A. niger* another pathway to deal with furfural.

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/fec-figur-1-mean-wt-2.png)
 *Figure 1: Cultivation of WT *A. niger* in YPD media with differing concentrations of furfural. Lines are mean values of triplicates and shadow marks +/- standard deviation of those.
Left) linear plot, Right) semilog plot. As can be seen on both subfigures, increasing concentration of furfural has a detrimental effect on the growth rate of WT *A. niger*
.*

### Characterising the effect of furfural on the growth of *A. niger* mutants

This cultivation was intervention styled: Spores were inoculated in minimal media (MM) with no furfural. After germination and when the cultures entered the exponential growth phase, furfural was added to a final concentration of: 0.0 g/l, 0.6 g/l, 1.2 g/l and 3.0 g/l. As can be seen in figure 2, hmfH mutant outgrew WT, while Arz mutant had similar growth curve until 60 hours of cultivation. However, increasing the concentrations of furfural impacted both the growth rate and final biomass gain of the mutants comapred to WT.

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/fec-figur-2-mean-0-0-gl-furfural.png)
![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/fec-figur-2-mean-0-6-gl-furfural.png)

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/fec-figur-2-mean-1-2-gl-furfural.png)
![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/fec-figur-2-mean-3-0-gl-furfural.png)
 *Figure 2: WT, Arz and hmfH mutants grown in MM with differing concentrations of furfural. Lines are mean values of triplicates and shadow is +/- the standard deviation of those. With no furfural, hmfH and to a certain extend Arz mutants grew better than WT, but with furfural added WT grew quite a bit better.*

After this cultivation, samples were processed and sent to our high-performance liquid chromatography (HPLC) lab for furfural quantification. With this, we could hopefully describe what could not be seen on the growth curves.
However, it was not possible to identify the furfural peak in the data from HPLC and subsequent analysis was thus not performed.

### Characterization of furfurals effect on the germination of *A. niger* spores

With the intervention styled experiment done, we wanted to see the effect of furfural on the germination of spores. Spores were added to MM with differing concentrations of furfural. After about 15 hour, WT spores in MM with no furfural began to germinate and Arz mutant spores germinated similarly compared to WT. Arz mutant spores only germinated slower than WT in low concentration of furfural (0.6 g/l). hmfH mutant spores were in general quite dealyed compared to WT.

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/fec-figur-3-mean-0-0-gl-furfural.png)
![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/fec-figur-3-mean-0-6-gl-furfural.png)

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/fec-figur-3-mean-1-2-gl-furfural.png)
![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/fec-figur-3-mean-3-0-gl-furfural.png)
 *Figure 3: WT, Arz and hmfH mutants grown in MM with differing concentrations of furfural. Lines are mean values of triplicates and shadow is +/- the standard deviation of those. Arz mutant germinated either similarly or a bit delayed depending on furfural concentration. hmfH mutant germination was also quite delayed compared to WT.*

## Menu

[Introduction](#Header3)
[Results](#Header4)

## Introduction

Engineered *A. niger* strains transiently expressing mCherry under the regulation of the fungal promoters scFDH1, BBa\_K4129004; anFDH, BBa\_K4129005; or scFDH2, BBa\_K4129006 were used to investigate the activity of the promoters and their response to furfural. Strains that carried only the empty vector (BBa\_K4129025) or mCherry cassette with a prokaryotic promoter (BBa\_K3046009) were used as negative controls, while strains carrying a constitutively expressed mCherry cassette (BBa\_K3046004) either on a plasmid or as a genomic insertion were selected as positive controls. The strains were grown in liquid minimal media. Half of the samples were induced with furfural either 20 or 70 hours after inoculation. Fluorescence and growth were monitored with a plate reader to evaluate promoter activity.
The anFDH promoter was further investigated by qPCR. The strain transiently expressing BBa\_K4129005 and a WT were grown on liquid minimal media, after 24 hours half the samples were induced with furfural. The FDH and mCherry transcripts were quantified at 1.5 hours following induction.

## Results

The strains utilising the investigated fungal promoters did not show a significantly higher fluorescence per biomass compared to the negative controls (Kruskal – Wallis, α = 0.05) (Figure 1), suggesting that, in *A. niger*, these promoters are neither constitutively active nor inducible in response to furfural. 1.5 hours after induction (Figure 1 D), many samples containing furfural showed an increase in fluorescence; however, this was also observed in the negative controls, suggesting that the increased fluorescence is due to a perturbation caused by furfural rather than a change in promoter activity.

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/results-fungal-promoters-fig1-a.png)
![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/results-fungal-promoters-fig1-b.png)

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/results-fungal-promoters-fig1-c.png)
![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/results-fungal-promoters-fig1-d.png)

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/results-fungal-promoters-fig1-e.png)
![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/results-fungal-promoters-fig1-f.png)
 *Figure 1. Comparison of the samples with or without furfural using fluorescence per biomass as a proxy for promoter activity. The bars indicate the mean fluorescence per biomass, the error bars correspond to +/-1 standard error. A-C shows the fluorescence per biomass of the samples induced at 20 hours after inoculation, as measured at 71.5, 94, or 118 hours, respectively. D-F shows the fluorescence per biomass of the samples induced at 70 hours after inoculation, as measured at 71, 94, or 118 hours, respectively.*

The qPCR results show that the FDH gene is strongly upregulated in response to furfural (Mann-Whitney test, α = 0.05) (Fig. 2 A). A similar trend was observed for the mCherry mRNA level (Fig. 2 B), however this difference was not supported by our statistical analysis (Mann-Whitney test, p-value = 0.1). We suspect that this discrepancy is caused by the small sample size at our disposal for the mCherry transcript, as the data from the WT strain could not be included in the mCherry qPCR, while both WT and BBa\_K4129005-expressing strains were included in the FDH mRNA quantification. This should be studied further, but until new data are collected we will simply suggest that BBa\_K4129005 is likely induced by furfural when transiently expressed in *A. niger*, and clearly upregulated when genomically integrated..

![Graph of relative mCherry expression 1.5 hours after induction](https://static.igem.wiki/teams/4129/wiki/results/results-fungal-promoters-fig2-a.png)
![Graph of relative mCherry expression 1.5 hours after induction](https://static.igem.wiki/teams/4129/wiki/results/results-fungal-promoters-fig2-b.png)
 *Figure 2. Investigation of anFDH promoter activity in response to furfural. The plots show the mRNA levels relative to actin mRNA of FDH (A), and mCherry transiently expressed from BBa\_K4129005 (B), as measured by qPCR.*

While the anFDH promoter shows strong induction both from the FDH locus and the plasmid vector, the absolute expression is clearly higher when measuring the FDH transcript. At the FDH locus, the promoter is integrated in a chromosome and in its native regulatory environment. While in the mCherry reporter, it is limited to 1000 bp and found on a vector for transient expression. We see two possible explanations for the observed difference in expression: either the anFDH regulatory regulatory region contains an enhancer that is not included in the 1000 bp we amplified, or the transient expression system is less efficient than the genomic locus. The latter is more likely; transient expression systems in filamentous fungi are unreliable, as can be observed in the fluorescence experiment where the positive control BBa\_K3046004 showed higher and more consistent fluorescence when genomically integrated rather than expressed transiently (Fig.1). To date,the anFDH promoter is the only *A. niger* promoter to be shown to be responsive to furfural. At the FDH locus the promoter has a considerable dynamic range and high activity, making it ideal for engineering a biosensor. Once genomically integrated, BBa\_K4129005 will be the first furfural biosensor ever built.

## Menu

[Introduction](#Header6)
[Results](#Header7)
[Characterization on solid media](#Header8)
[Characterization in liquid culture](#Header9)

## Introduction

The fungal synthetic expression system (SES) constructed in this project consists of our synthetic transcription factor (sTF), synthetic minimal promoter (6xLexO-Pmin) and mCherry. The sTF is constitutively expressed from PgpdA and it is expected that the sTF promotes transcription of the mCherry.

We successfully assembled 27 SESs each with their unique sTF, and 12 of these had their functionality tested in *A. niger*, both grown on solid media and in liquid media, by measuring fluorescence.
The solid media experiments were carried out in two batches and there were minor differences in execution of the assessment. The liquid media experiments were measured with the same gain across multiple plates in order to compare fluorescence.

The 12 SESs were tested and a few SESs were highlighted to give a quick overview of some results. The sTF of the SESs were FunsTF02, FunsTF04, FunsTF05, FunsTF18, FunsTF18, FunsTF57 and FunsTF70. All of the sTF used lexA as a DNA-binding domain. FunsTF04 and FunsTF05 have Hmox1 as ligand-sensing domain, while FunsTF02 has HbaR. FunsTF18, FunsTF57 and FunsTF70 have mutated variants of HbaR. The transactivation domain B112 is used in FunsTF02, FunsTF04 and FunsTF18, while FunsTF05, FunsTF57 and FunsTF70 have VP16.

*Table 1: An overview of floursences results of the following highlighted sTFs. They are FunsTF02, FunsTF04, FunsTF05, FunsTF18, FunsTF57 and FunsTF70. The sTFs were tested in minimal media, MM with 2 mM benzoic acid and MM with 0.6 g/L furfural. The results are illustrated relative to the best performing SES of FunsTF05.*
![Overview of FunsTFs results.](https://static.igem.wiki/teams/4129/wiki/results/overview-of-ses.png)

## Results

This chain of experiments did not supply us with the groundbreaking results we had hoped for, but will provide a good starting point for further work, since there are only a handful of synthetic systems that function in filamentous fungi. All SESs documented to work in filamentous fungi until now are constitutive systems, making this SES the first documented inducible SES in filamentous fungi. sThose systems do not have the possibility to become inducible. They were created to be constitutive.

## Characterization on solid media

This chain of experiments did not supply us with the groundbreaking results we had hoped for, but will provide a good starting point for further work, since there are only a handful of synthetic systems that function in filamentous fungi. All SESs documented to work in filamentous fungi until now are constitutive systems, making this SES the first documented inducible SES in filamentous fungi. Those systems do not have the possibility to become inducible like our systems.

![Overview of FunsTFs results.](https://static.igem.wiki/teams/4129/wiki/results/ses-control-et104.jpg)
 *Figure 1: Pictures of fluorescence from *A. niger*, which carries either BBa\_K4129025 or genomically integrated BBa\_K3046004. The exposure time was set to 1.04 seconds. The *A. niger* is grown on plates containing minimal media, minimal media with 2 mM benzoic acid or minimal media with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity.*

The first iteration had an exposure time of 1.04 seconds and the genomic integrated BBa\_K3046004 and BBa\_K4129025 showed the expected outcome. BBa\_K4129025 is an empty vector, thus it did not account for the potential expression leak from 6xLexA-Pmin (BBa\_K4129115)

FunsTF04 and FunsTF05 have a similar structure but the transactivation domain was B112 and VP16 for FunsTF04 and FunsTF05, respectively.

FunsTF05 displays higher intensity than genomic integrated BBa\_K3046004. A contributing factor, to this observation, was that FunsTF05 was carried on a high copy transient expression plasmid. The genomic integrated BBa\_K3046004 was integrated as a single copy (figure 1). FunsTF04 displays only fluorescence at the perimeter of *A. niger* when grown on minimal media (figure 1).
The fluorescence from FunsTF04 has a higher intensity than that of BBa\_K4129025. It can be argued that the fluorescence was due to leaky expression from 6xLexA-Pmin (BBa\_K4129115). The leaky expression does not fit because FunsTF04 on benzoic acid does not express, thus it indicates there is no leak or working sTF (figure 2).
To summarise, The sTF of FunsTF05 promotes constitutive expression of mCherry. In addition, the activity of FunsTF04 was not at the same level as FunsTF05. Which indicates that VP16 was a more active transactivation domain than B112, in this specific combination of modules (LexA, Hmox1, and SV40).

![Overview of FunsTFs results.](https://static.igem.wiki/teams/4129/wiki/results/ses-funstf0405-et104.jpg)
 *Figure 2: Pictures of fluorescence from *A. niger*, which carries either FunsTF04 or genomically integrated FunsTF05. The exposure time was set to 1.04 seconds. The *A. niger* is grown on plates containing minimal media, minimal media with 2 mM benzoic acid or minimal media with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity.*

The SES with FunsTF02 (BBa\_K4129101) also displays slight fluorescence at the perimeter of the colonies when grown on minimal media. This fluorescence was not present on any other MM plates with either benzoic acid or furfural (figure 3).FunsTF02 is interesting because it is the sTF, which is closest to sBAD (Castaño-Cerezo et. al (2020)). A sTF that was inducible in S. cerevisiae, is working in *A. niger*, but not it is not inducible yet.
FunsTF57 did not display any fluorescence even when compared to BBa\_K4129025 across all three different medias (figure 3).

![Overview of FunsTFs results.](https://static.igem.wiki/teams/4129/wiki/results/ses-funstf0257-et104.jpg)
 *Figure 3: Pictures of fluorescence from *A. niger*, which carries either FunsTF02or genomically integrated FunsTF57. The exposure time was set to 1.04 seconds. The *A. niger* is grown on plates containing minimal media, minimal media with 2 mM benzoic acid or minimal media with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity.*

The second iteration used an exposure time of 0.72 seconds. At this exposure time FunsTF18 and FunsTF70 were constitutively expressing mCherry, although it was with mid intensity. FunsTF18 had the modules as FunsTF02, but the HbaR domain of FunsTF18 was our mutation 12. FunsTF18 was observed to be constitutively, so another set of mutations to HbaR is required to improve inducibility.

![Overview of FunsTFs results.](https://static.igem.wiki/teams/4129/wiki/results/ses-funstf1870-et072.jpg)
 *Figure 4: Pictures of fluorescence from *A. niger*, which carries either FunsTF18or genomically integrated FunsTF70. The exposure time was set to 0.72 seconds. The *A. niger* is grown on plates containing minimal media, minimal media with 2 mM benzoic acid or minimal media with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity.*

The remaining SESs did not show any fluorescence and all pictures can be found here (link to [Download](https://static.igem.wiki/teams/4129/wiki/results/sup-plates.pdf) PDF with all pictures).

## Characterization in liquid culture

The *A. niger* expressing our SESs were cultivated in a 48 well CellStar cultivation plate and 24 hours after inoculation the SES systems were induced. The inducers were 2 mM benzoic acid and 0.6 g/L furfural. The fluorescence and optical density at 600 nm (OD600) was measured at four time points: 2 hours, 26, hours, 47 hours and 70 hours after induction. The measurements were performed in a CLARIOstar microplate reader.

The genomic integrated BBa\_K3046004 was used as positive control and pAC572 (BBa\_K4129025 ) was used as a negative control. A clear inducibility of the sTFs was not observed, because the samples induced with 2 mM benzoic acid or 0.6 g/L furfural did not fluoresce more than the uninduced samples (figure 5).

Two hours after induction, the observed fluorescence for the sTFs were slightly higher than that of pAC572. This was supported by the sTFs are all significantly different than pAC572 (Kruskal - Wallis, α=0.05)
FunsTF05, while uninduced, had the highest fluorescence of all the sTF at around 3300 RFU (figure xx). FunsTF02 induced with 2 mM benzoic acid did also have a high fluorescence a little under 3000 RFU, but it was not significantly different from pAC572 induced with 2 mM benzoic acid (Kruskal - Wallis, α=0.05).

The time points of 26 hours, 47 hours and 70 hours after induction displayed similar observations of fluorescence measurement, The main point of interest is that FunsTF05 has high activity throughout time and FunsTF18 ramps up slowly over time.

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/mcherry-expression-selstrains-2h-g1500-bar.png)
![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/mcherry-expression-selstrains-26h-g1500-bar.png)

![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/mcherry-expression-selstrains-47h-g1500-bar.png)
![Plasmid map](https://static.igem.wiki/teams/4129/wiki/results/mcherry-expression-selstrains-70h-g1500-bar.png)
 *Figure 5: Barplot of log transformed fluorescence (RFU) with standard deviation displayed as arrows. The fluorescence measurements from *A. niger* carrying genomically integrated BBa\_K3046004, BBa\_K4129025, FunsTF02, FunsTF04, FunsTF05, FunsTF18, FunsTF57 and FunsTF70. The *A. niger* was grown in minimal media, where it was uninduced (blue), induced with 0.6 g/L furfural (red) or induced with 2 mM benzoic acid (green). The fluorescence was measured A) 2 hours, B) 26 hours, C) 47 hours, and D) 70 hours after induction.*

Supplementary data - [Download](https://static.igem.wiki/teams/4129/wiki/results/sup-liquid-testing.pdf)

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