iGEM 2022 — Team thessaly

{% extends "layout.html" %}
{% 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 %}
{% block page\_content %}

## What should this page contain?

---

* 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

---

* [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)

{% endblock %}

{% extends "layout.html" %}
{% 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 %}
{% block page\_content %}

Overview

Synthetic or Engineering Biology is a field of science that involves redesigning organisms for useful purposes by engineering
them to have new abilities. By breaking the problem apart into smaller ones and following iterations of the DBTL cycle (Design,
Build, Test, Learn) to focus individually on each one, we can simplify the development of **a functional complex biological system**.
So with the goal of eutrophicated water bioremediation, we engineered a device that could help absorb more nutrients in the presence
of eutrophication. Moving down the abstraction hierarchy, we came up with a 2-module system; a detection module and a nutrient
uptake module. The first consists of the **TetR-KRAB silencing complex**, which is under the regulation of **a synthetic riboswitch**, able
to detect the presence of **microcystin-LR (biomarker)**. The second module is a TetR-regulated **PHT1 transporter overexpression cassette**.

![Representation of our synthetic device in the absence (left) and presence (right) of microcystin-LR.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/constructsfinal.png)

**Figure 1.** Representation of our synthetic device in the absence (left) and presence (right) of microcystin-LR.

Here we illustrate the characterization of both of these constructs by performing 3 different DBTL cycles, with several iterations.

![Iterative Design-Build-Test-Learn (DBTL) cycle of our Engineering Cycles](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/engin.png)

**Figure 2.** Iterative Design-Build-Test-Learn (DBTL) cycle of our Engineering Cycles

Engineering Cycle 1: Our Riboswitch Engineering (Construct 1)

Our goal was to design **a microcystin-LR (MC-LR) responsive, synthetic riboswitch**. This is a process that usually involves in
silico engineering, ***in vitro*** and/or ***in vivo*** testing of final molecules. Given the narrow time frame and limited resources we
had to conduct experiments, we nonetheless managed to establish the computational design. Despite the scarcity of tools available
for RNA riboswitch engineering (compared to protein engineering), we have successfully demonstrated that this pipeline can produce
theoretically verified RNA receptor – ligand couples for any small molecule, if an aptamer is at hand.
This work required several iterations of the Design/Build – Test – Learn cycle. We started with the sequences of **the plant thiamine
pyrophosphate (TPP) riboswitch and the microcystin-LR aptamer**, combined them to yield hundreds of random sequences to choose from,
screened the candidates according to secondary and tertiary structure formation, 3’ splicing site functionality and successful ligand
docking. During this process, at multiple occasions, **we had to take a step back to correct and optimize the pipeline, thus ending up with
six DBTL cycle iterations.**

#### First Iteration: Studying the TPP riboswitch

##### Design & Build

To achieve sensing of exogenous signals by plants, one can think of many ways to engineer them. We went with the design of
a new riboswitch because a MC-LR targeting aptamer was described in a recent study[1](#references). We chose the plant TPP riboswitch as
a backbone, as it is the only riboswitch present in plants[2](#references). Another advantage was the size of the ligands. TPP (MW of 424, 31)
[3](#references) and MC-LR (MW of 995,2) [4](#references) are far more comparable than MC-LR and other small molecules targeted by bacterial riboswitches,
like theophylline [5](#references) or adenine [6](#references). To ensure that the secondary and tertiary structure prediction methods we would use could provide
as accurate results as possible, we tested them with the sequence of the TPP riboswitch [7](#references) and compared the result with the
experimentally determined 3D structure.

##### Test

The .fasta and .pdb files from RCSB PDB (id: 3D2G) were obtained and the .fasta file was fed into the RNAfold [8](#references) algorithm
to produce a secondary structure prediction. The result was given to the RNAcomposer program [9](#references) to calculate the tertiary
structure. The final .pdb file was compared with that of 3D2G.

##### Learn

About 35% (including the part where TPP binds to) of the prediction was identical to 3D2G, with an alignment from RNA-align [10](#references).
We now know that a combination of RNAfold and RNAcomposer can produce accurate enough predictions for our purpose.

![Comparison of the TPP riboswitch RNAcomposer - determined 3D structure and the experimentally calculated one.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/eng3.png)

**Figure 3.** Comparison of the TPP riboswitch RNAcomposer - determined 3D structure and the experimentally calculated one.

#### Second Iteration: Designing the new riboswitch – Secondary Structure (First try)

##### Design & Build

Not only is the TPP riboswitch special in that it is present in eukaryotes, but contrary to other switches, it functions by
affecting intron splicing in pre-mRNA [11](#references). When TPP binds to its aptamer, it promotes the formation of a long and unstable mRNA,
by causing a poly-A signal to be spliced out of the 3’ UTR. TPP aptamer folding, prior to ligand binding, includes both 5’ and 3’
splicing-site (SS) sequestering elements, by the secondary and tertiary structure respectively, with further complex structural
rearrangements occurring upon binding [12](#references). With this level of complexity, splicing regulation through this particular mechanism appears
to be far from amenable to rational replication with simple bioinformatic tools. Our approach, then, was confined to the replacement of
the TPP aptamer with the MC-LR one, to achieve 3’ (SS) sequestration by its tertiary structure. Similar attempts have been reported in
the past with the Tetracycline Repressor (TetR) protein and theophylline aptamers [13](#references), [14](#references) in mammalian cells, but never in plants.

##### Test

The randomseq.py program [15](#references) was used to calculate multiple random sequences of overhangs for the MC-LR aptamer. These are small
sequences that will be flanking the aptamer region inside the riboswitch, to “protect” it from unpredictable structural arrangements.
Hence, we paid close attention to this RNA element’s modularity prospects, one of the foundational ideas of Synthetic Biology. The outcome
of this algorithm was well over a thousand sequences. These were printed in a .txt file which was used as an input for RNAfold.

```
randomspythoneq.py MS ‒n=1000 ‒cap_start=False ‒cap_stop=False
‒fasta=True ‒prefix=’Test’ ‒statement=’v(15,15,False,False,False,False);
c (GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCGC);
V (20,20,False,False,False,False)’
> results.txt
```

##### Learn

The result was a file with pictures of these sequences’ secondary structure. A quick look over them was enough to see that most
were obviously problematic and could not be used, because they had no structural elements (like loops and junctions) for the
tertiary structure to form.

![Examples of problematic secondary structures.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/defect-secondary.png)

**Figure 4.** Examples of problematic secondary structures.

#### Third Iteration: Designing the new riboswitch – Secondary Structure (Second try)

##### Design & Build

According to the manual pages of RNAfold, to force constraints on the algorithm, one must manually create a file with the
nucleotides that should be paired or unpaired, or create loops etc. To do that, a clear picture of the aptamer’s 3D structure
is required. This was calculated with the iFoldRNA web server [16](#references), and the .pdb files were visualized with UCSF Chimera [17](#references).
Nucleotides positioned both in the middle of the sequence and inside “pockets” of the structure, were used to apply constraints [18](#references).

![The aptamer domains that were used to apply folding constraints. Created with Molstar Viewer.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/eng5.png)

**Figure 5.** The aptamer domains that were used to apply folding constraints. Created with Molstar Viewer.

##### Test

The algorithm ran again with the constraint file.

```
RNAfold –constraint[=constraint.txt] > sec_str_corrected.fasta
```

##### Learn

Indeed, most secondary structures now looked better and could be used for the next step.

#### Fourth Iteration: 3’ Splicing Site Functionality test

##### Design & Build

The .fasta file of the *A.thaliana* thiC gene 3’ UTR (where the TPP riboswitch naturally occurs) was obtained
(NCBI Reference Sequence: NM\_001336219.1). It was time to replace the TPP aptamer with the MC-LR aptamer candidates. The whole
scheme now should work as a normal intron, with a 5’ SS and a branch point site from the natural sequence, and a 3’ SS from the
new aptamer.

##### Test

To test that, some sequences were randomly selected to look for splicing sites by the NetGene2.0 web server [19](#references). Only one of
them yielded a satisfying result. This meant that at least a few of the sequences were appropriate and now they had to be found
among the hundreds. A simple bash script utilizing the grep command and regular expressions targeting the 3’ SS consensus sequence
for *A.thaliana* [20](#references), enabled precise selection of the best candidates. NetGene2.0 ran again with these sequences, and all worked as
expected.

```
grep -E ‘Τ(A|G|Τ)CAG(G|Α)(A|Τ)’ results.txt > new_results.txt
```

##### Learn

Starting with a thousand sequences, we were now **left with 15 that could be used for 3D structure prediction**.

#### Fifth Iteration: Designing the new riboswitch – Tertiary Structure

##### Design & Build

While secondary structure calculation is a crucial step for the functional characterization of an RNA molecule, it is the
three-dimensional structure that determines interactions with ions and ligands inside the cell. For this purpose, the 15 2D
structures from the previous step were used as input for RNAcomposer. Our goal was to produce 3D structures with a good RMSD
score for the docking step. The structures were also compared with the one predicted with iFoldRNA (see 3rd iteration).

##### Test

RNAcomposer ran, and the output files were tested with RNAlyser [21](#references), to check for RMSD and similarity with the iFoldRNA predicted
.pdb file.

##### Learn

As a result of this screening, 6 of the structures made it to the final step; docking.

#### Sixth Iteration: Docking

##### Design & Build

Our goal in this final step was to obtain a secondary structure, to which MC-LR could bind in a reasonable position. There are
very few options for tools to perform in silico RNA-ligand docking experiments. Whereas we could use common docking applications
like AutoDock Vina, ZDOCK or DOCK 6.5, these are better suited for protein design. With further research, we found RLDOCK [22](#references) and
AnnapuRNA [23](#references). These are RNA specific tools, utilizing scoring functions to predict RNA-small molecule interactions. Because RLDOCK
needed too much computational power, something we did not possess (at least 64 GB RAM), we went with AnnapuRNA.

##### Test

The receptor (RNA) and ligand (MC-LR) PDB files were prepared for the program to run.

```
./annapurna.py -r cluster1.pdb -l MCLR_ligand.titles.sdf -m kNN_basic -m kNN_modern -o testresults/output  -s --overwrite --groupby --merge --cluster_fraction 1.0 --cluster_cutoff 2.0 --clustering_method AD
```

##### Learn

At least half of our sequences bind the ligand in a protected “socket”, created by the aptamer and appear to be suitable for use as
a new, MC-LR responsive riboswitch. This would be a 3’ UTR Phytobrick with B6-C1 overhangs [24](#references). However, the functionality of such a
system remains to be validated with in vitro and, most importantly, in vivo experiments in plants. We hope that we have inspired future
iGEM teams to follow a similar process for synthetic splicing riboswitch design or even test our own.

![Cartoon of the aptamer - microcystin docking that was calculated with AnnapuRNA. Created with Molstar Viewer.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/eng6.png)

**Figure 6.** Cartoon of the aptamer - microcystin docking that was calculated with AnnapuRNA. Created with Molstar Viewer.

Engineering Cycle 2: Our Repressor Engineering (Construct 1)

Repressors have been widely used in synthetic biology as they allow for a precise control of gene expression, producing a desired
phenotype. Amid the field, are repressors that can function properly in plants and amongst the most prevalent ones are the Tet
Repressors [25](#references), as was suggested to us by [Prof. Kalliope Papadopoulou](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27human-practices%27%29%20%7D%7D). The registry of biological parts already provided a standardized
part ([BBa\_C0040](http://parts.igem.org/Part%3ABBa_C0040)) with the coding region for the TetR protein without a Ribosome Binding Site (RBS) so we based our design on this sequence.
Before we continued with the TetR design, we needed first to establish the basic parts of our transcriptional unit and test their functionality.

![Design of a Transcriptional Unit; Cloning Overhangs, Promoter, 5'UTR, Coding Sequence, 3' UTR, terminator respectively](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/upomnimateliko.png)

**Figure 7.** Design of a Transcriptional Unit; Cloning Overhangs, Promoter, 5'UTR, Coding Sequence, 3' UTR, terminator respectively.

#### First Iteration: Deciding on Regulatory elements

##### Design

###### The Reporter System

Starting with the promoter of choice, we opted for the **Nopaline Synthase promoter (NOS promoter)** as it is a promoter proven to work
in plants [26](#references) and a standardized Phytobrick [27](#references) that is weaker than the obvious counterpart (CaMV 35s promoter) [28](#references). Lower expression
meant less possibility for toxicity symptoms caused by high expression of TetR [29](#references) and for better responsiveness of the system to
tetracycline. Continuing with the reporter system, **mVenus** [30](#references) was the protein of choice as this is an autofluorescent protein
(AFP) (Excitation λ: 515 nm, Emission λ: 527 nm, 31) that is well characterized in Nicotiana benthamiana. We paid careful attention
to the **Kozak frame** of the protein and given that, in green plants, a characteristic motif has been observed
$$R\_{(-3)} \;M\_{(-2)}\; –\_{(+1)}\; A\_{(+1)} \;U\_{(+2)} G\_{(+3)} G\_{(+4)} C\_{(+5)} $$[32](#references), we made two substitutions (4:T>C, 5:G>T) to the ORF leading to a mutation (V2A) in the amino acid (aa)
sequence, which should not cause folding and activity mishaps due to the position and size of the two aa. Lastly, what remains is the
terminator. Settling, again, on **Nopaline Synthase (NOS terminator)**, this is a strong terminator that is a standardized Golden Braid
part [33](#references). Putting it all together, we created a promising Reporter System, consisting of pNOS:mVenus:tNOS ([BBa\_K4213029](http://parts.igem.org/Part%3ABBa_K4213029)).

##### Experimental Design-Plant Selection

While our end-product was already decided to be the plant *P. australis*, we needed a stepping stone, a model plant to perform
the first characterization and optimization of our constructs. For this, we selected *Ν. benthamiana* since it allows for high and
rapid expression of transgenes, by agroinfiltration, as the species is quite susceptible to plant viral vectors [34](#references). *N. benthamiana*
leaf samples are also able to be observed under UV lumination for expression of fluorescent proteins [34](#references).

##### Experimental Design-Cloning method

Among the prevalent cloning methods used today, one that stands behind its simplicity, effectiveness and rapidness is the Golden Braid
method [33](#references). Based on the Golden Gate system, this technology relies on the Type IIS restriction enzymes to alternate between level
Alpha and Omega states, resulting in the assembly of one or more Transcriptional Units (TUs) One of the main benefits of Golden Braid
is the four nucleotide cutting sequence of the restriction enzymes, which can be completely random, equipping it with the ability to
completely fix the order of insertion between different parts by designing different complementary four-nucleotide overhangs at each end.
For our assembly, we used the proposed sequences of the technique.

![Proposed overhang sequences of the Golden Braid method for the different parts of a TU (A1-C1)](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/trans.png)

**Figure 8.** Proposed overhang sequences of the Golden Braid method for the different parts of a TU (A1-C1).

##### Build

Next came the cloning experiments that lead to the assembly of our Reporter System. The procedure was conducted on a special strain
of *E. coli*, K-12 DH5a, engineered to maximize transformation efficiency. With the help of diagnostic digestion and analysis by gel
electrophoresis we were able to confirm the assembly of our construct.

![Diagnostic Digestion of pDGB3α2_pNOS-Venus-tNOS with HindIII and expected bands (bp): 6345, 1033 and 509.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/1.png)

**Figure 9.** Diagnostic Digestion of pDGB3α2\_pNOS-Venus-tNOS with HindIII and expected bands (bp): 6345, 1033 and 509.

##### Test

As mentioned above, we selected *N. benthamiana* for our experiments, so what followed was the transient expression of our constructs
*in planta* through agroinfiltration. This method allows for noticeable expression of transgenes within 4 days of infiltration[39](#references)
and, with the fluorescent mVenus, we planned to observe transgene expression under UV luminescence using fluorescent microscopy.

![Final Steps to insert our built constructs and observe their Fluorescence](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/steps2.png)

**Figure 10.** Final Steps to insert our built constructs and observe their Fluorescence.

##### Learn

After examining our samples under the confocal microscope and taking our pictures (Figure 11) we concluded that our transcriptional
unit did manage to express mVenus in great numbers and, therefore, we moved on to the second iteration of this DBTL cycle, to test
our design with the repressor.

![Agroinfiltration experiment: positive (pDGB3α2_pNOS-Venus-tNOS) control. The red arrows show intercellular bridges that confirm that Venus protein is in the cytoplasm.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/2.png)

**Figure 11.** Agroinfiltration experiment: positive (pDGB3α2\_pNOS-Venus-tNOS) control. The red arrows show intercellular bridges that confirm that Venus protein is in the cytoplasm.

#### Second Iteration: Establishing Repressor Expression

##### Design

Having learnt that the above-mentioned syntax works as intended, it was time to substitute the fluorescent protein with our repressor
of choice. As already mentioned, the registry gave as the base sequence for TetR (BBa\_C0040), but this part was intended for expression
in prokaryotic organisms and not eukaryotic ones. For that reason, we needed to anticipate the presence of a nucleus, possible splicing
sites and unoptimized expression on account of less frequently used synonymous codons present in the coding sequence (cds).

###### Tet Repressor for plants

Starting with the first, it seemed that due to the size of TetR (48 kDa) diffusion into the cell nucleus was more than likely [40](#references).
Nevertheless, we decided to add a Nuclear Localization Signal (NLS), specifically Simian Virus 40 NLS [41](#references), to the sequence since this
would ensure its passage into the nucleus. Moving on to the possible splicing sites, the now NLS-containing sequence was run through a
neural network based program called NetGene2. This method is proven to predict possible splicing sites within given sequences, based
on analysis made on the plant *Arabidopsis thaliana* [42](#references). Analysis showed no potential splicing sites within the cds so we moved on with
no modifications. Concluding with codon optimization, this is a tool revolving around the frequency that each synonymous codon occurs
within a certain organism’s genome. We used IDT’s codon optimization tool for expression in *Nicotiana benthamiana*.
It is worth pointing out that, for the visualization of expression, we, again, decided to incorporate the modified mVenus into the sequence,
leading to a construct consisting of **pNOS:Venus:TetR:NLS:tNOS ([BBa\_K4213030](http://parts.igem.org/Part%3ABBa_K4213030))**.

##### Build

Following the same steps as before and making the necessary optimizations to our protocols, needed to properly and efficiently get
through the cloning procedures, we managed to confirm the assembly of the level alpha construct using diagnostic digestion (Figure 12).

![Diagnostic Digestion of pDGB3α2_pNOS-Venus-TetR-tNOS with HindIII and expected bands (bp): 6345, 1678 and 513.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/3.png)

**Figure 12.** Diagnostic Digestion of pDGB3α2\_pNOS-Venus-TetR-tNOS with HindIII and expected bands (bp): 6345, 1678 and 513.

##### Test

The testing of the produced construct was, again, done with model plant *Nicotiana benthamiana* by agroinfiltration. The sample was prepared
with a special cell-permeant nuclear counterstain that emits blue fluorescence when bound to dsDNA (Hoechst 33342, Excitation λ: 350 nm,
Emission λ: 460 nm) and can be distinguished from mVenus, when observed under both fluorescent microscopy and confocal microscopy, in order
to confirm localization of our construct in the nucleus.

##### Learn

After examining our samples under the confocal microscope and taking our pictures we concluded that our transcriptional unit did
manage to express mVenus:TetR:NLS in great numbers and the fused protein was mostly present within the nucleus, confirming the
functionality of the NLS (Figure 13).

![Agroinfiltration experiment: pNOS-TetR-tNOS. The red arrows show the nuclei, where the Venus-TetR protein is located.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/4.png)

**Figure 13.** Agroinfiltration experiment: pNOS-TetR-tNOS. The red arrows show the nuclei, where the Venus-TetR protein is located.

#### Third Iteration: Upgrading repressing abilities

##### Design

It was pointed out to us by Prof. Aikaterini Kalliampakou that tetracycline-controlled systems can be quite leaky. After careful
literature review, we learnt that ‘leaky’ gene expression is a common and rather persistent problem in synthetic biology and often
contributes to the poor performance of a genetic circuit [43](#references). This led us to the investigation and utilization of the Tet toolbox [44](#references).

###### Improving our repressor with a transregulator

In more detail, we decided to use a part fitter for eukaryotic systems, the TetR-KRAB transregulator. As the name suggests,
TetR has been fused with the Krüppel Associated Box protein (KRAB) [45](#references), enhancing the inhibition abilities of the complex by
adding another mechanism of silencing [46](#references). Since our design already incorporated TetR, we just needed to add KRAB next to the
repressor as this is the supposed working syntax [47](#references). For this we had to remove the already present stop codon by inducing a mutation.
Furthermore, after the conducted optimization of the part, we had to domesticate it by inducing a silent mutation, in order to remove
an unwanted recognition site for BsaI.
The final syntax contained mVenus as, again, we wanted to spot protein expression with the help of fluorescent microscopy. This led
to a final construct containing **pNOS:Venus:TetR:NLS:KRAB:tNOS([BBa\_K4213033](http://parts.igem.org/Part%3ABBa_K4213033))**.

##### Build

After almost three weeks of cloning experiments, we managed to confirm the assembly of the abovementioned construct by diagnostic
digestion and visualization through gel electrophoresis.

![Diagnostic Digestion](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/5.png)

**Figure 14.**
Diagnostic Digestion of:
1. and 3 “pNOS-Venus-TetR-KRAB-tNOS“ with HindIII (expected bands in bp: 6345, 2056 and 513)
2. and 5 “pNOS-TetR-KRAB-tNOS“ with HindIII (expected bands in bp: 6345, 1342 and 513)
Positive results: 2, 4 and 5 1 MW.

##### Test

We repeated agroinfiltration in *Nicotiana benthamiana* leaves and our samples were prepared with the previously mentioned counterstain
for better visualization of the nucleus for observation under the confocal microscope.

##### Learn

With the images presented below, we confirm the expression of our transregulator as well as its localization in the nucleus.

![Agroinfiltration experiment: pNOS-Venus-TetR-KRAB-tNOS.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/6.png)

**Figure 15.** Agroinfiltration experiment: pNOS-Venus-TetR-KRAB-tNOS.

Engineering Cycle 3: Our PHT1 Engineering (Construct 2)

As mentioned above, the purpose of our entire synthetic system is the increased absorption of phosphorus, which will only be induced
by the presence of microcystin-LR in the roots of the plant. Now, we come to the second part of our system. This module consists of
a transcriptional unit that contains the *pht1* gene with a constitutive promoter, whose structure is engineered to be inhibited in the
presence of the TetR protein. So, again, we had to start with the basic parts of the system.

#### First Iteration: Deciding on Regulatory Elements

##### Design

###### The Second Reporter System

Beginning with the promoter, we opted for the Cauliflower Mosaic Virus 35S promoter (p35s) as this is arguably the most well-studied
and experimentally used regulatory component with activity in plant cells [48](#references). With abundant information available on its individual
functional domains, we could incorporate Tet Operator (TetO) sequences within the promoter, placing the second construct under the
influence of TetR. Therefore, we introduced three repeats of TetO to p35s and set them in close proximity to the TATA box, based on the
design of pTriple Op promoter [49](#references). Given that we planned to test both TetR and TetR-KRAB for their repressive abilities, we also introduced
seven direct repeats of the Tet Response Element (TRE) upstream of the distal part of p35s, based on the design of pTight (also known as
pTetO7)[50](#references). With the distance between the TIS and the modified TRE being under 3.000 bases, this was the best fit for TetR-KRAB’s long
range repressive power [50](#references). Continuing on, both the protein and the terminator of choice would be the same as the first reporter system
so we ended up with a final construct containing mod-p35s:Venus:tNOS ([BBa\_K4213041](http://parts.igem.org/Part%3ABBa_K4213041)).

##### Build

After two weeks of cloning experiments, we managed to confirm the assembly of the abovementioned construct by diagnostic digestion
and visualization through gel electrophoresis.

![Diagnostic Digestion of “TetO7-pTriple-Venus-tNOS(s)” with EcoRI and HindIII and expected bands in bp: 6345, 1829, 674 and 387.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/7.png)

**Figure 16.** Diagnostic Digestion of “TetO7-pTriple-Venus-tNOS(s)” with EcoRI and HindIII and expected bands in bp: 6345, 1829, 674 and 387.

##### Test

We repeated agroinfiltration in *Nicotiana benthamiana* leaves but this time we were not able to visualize our constructs in our University’s
confocal microscope, due to malfunctions. Fortunately, we were able to measure the fluorescence of our prepared samples with the help
of the Plate Reader.

##### Learn

With the results presented below we confirm the expression mVenus and, therefore, the functionality of our modified p35s. Next step
was the expression of the *pht1* gene.

![Fluorescence of different leaf samples measured under the Plate Reader.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/8.png)

**Figure 17.** Fluorescence of different leaf samples measured under the Plate Reader.

#### Second Iteration: Establishing Transporter Expression

##### Design

After the confirmation of expression, we could move on to our phosphorus transporter. We decided to test the genes of PHT1;5
from *Arabidopsis thaliana* [51](#references) [52](#references) and PHT1;6 from *Oryza sativa* [53](#references) [54](#references) Pi transporters due to their evolutionary relatedness of
each source organism to our end-product plant (P. australis) and their potential to be overexpressed without showing significant levels of
toxicity, leading to even higher Pi uptake regardless of its concentration [55](#references). So after finding the cds of each gene, we did two rounds
of modifications. Firstly, we searched for the same Kozak Frame motif around the TIS, leading to two substitutions in the OsPHT1;6 sequence
(+5: G>C, +6: A>T) and a mutation in the aa sequence (G2A) which should not cause a problem to the functionality of the transporter due to
the size and the place of the two aa. Secondly, we decided to test the effects of codon optimization on these transporters by ordering both
codon optimized and not. Sadly, the high complexity of the OsPHT1;6 sequence did not allow us to order it from neither IDT nor TWIST, therefore
we partially optimized the spots characterized as “highly complex” by the tool. Finally, as again we planned to visualize expression
through fluorescent microscopy, we included a linker sequence and our reference gene (mVenus) downstream of the part. To summarize, our
final versions of construct 2 were:

1. mod-p35s:(optimized)AtPHT1;5:Venus:tNOS ([BBa\_K4213035)](http://parts.igem.org/Part%3ABBa_K4213035)
2. mod-p35s:(non-optimized)AtPHT1;5:Venus:tNOS ([BBa\_K4213036](http://parts.igem.org/Part%3ABBa_K4213036))
3. mod-p35s:(optimized)OsPHT1;6:Venus:tNOS ([BBa\_K4213040](http://parts.igem.org/Part%3ABBa_K4213040))
4. mod-p35s:(partially-optimized)OsPHT1;6:Venus:tNOS ([BBa\_K4213043](http://parts.igem.org/Part%3ABBa_K4213043))

##### Build

Repeating the same steps and after several weeks of cloning, together with sequencing, we managed to confirm the assembly of
the abovementioned constructs.

![Diagnostic Digestion.](https://static.igem.wiki/teams/4213/wiki/wiki/img/wet-lab/engineering/9.png)

**Figure 18.**
Diagnostic Digestion of:
1. 2 and 3 “TetO7-pTriple-AtPHT1;5 opt-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 1978, 1492, 674 and 387)
2. 4 and 5 “TetO7-pTriple-AtPHT1;5 non-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 1978, 908, 674, 584 and 387)
3. 6 and 7 “TetO7-pTriple-OsPHT1;6 opt-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 2041, 1405, 674 and 387)
4. 8 and 9 “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 3449, 674 and 387)
Positive results: 2, 4 and 5 1 MW.

##### Test

Again, after agroinfiltration and preparation of samples like before, we tried to observe our constructs under the confocal microscope.

##### Learn

Unfortunately, we encountered a problem with the setup of our confocal microscope and due to the time constraints we were not able
to rerun the experiment in order to confirm the expression and localization of the transporters.

Concluding

After 3 different DBTL cycles, with several iterations, we can present to you our two new and optimized expression platforms
for *N.benthamiana* ([BBa\_K4213029](http://parts.igem.org/Part%3ABBa_K4213029), [BBa\_K4213041](http://parts.igem.org/Part%3ABBa_K4213041)) and 2 optimized versions of TetR for expression in *N.benthamiana* ([BBa\_K4213044](http://parts.igem.org/Part%3ABBa_K4213044), [BBa\_K4213045](http://parts.igem.org/Part%3ABBa_K4213045)).

### References

1. Ng, A., Chinnappan, R., Eissa, S., Liu, H., Tlili, C., & Zourob, M. (2012). Selection, characterization, and biosensing application of high affinity congener-specific microcystin-targeting aptamers. Environmental science & technology, 46(19), 10697–10703
2. Bocobza, S., Adato, A., Mandel, T., Shapira, M., Nudler, E., & Aharoni, A. (2007). Riboswitch-dependent gene regulation and its evolution in the plant kingdom. Genes & development, 21(22), 2874–2879.
3. <https://pubchem.ncbi.nlm.nih.gov/compound/Thiamin-pyrophosphate>
4. <https://pubchem.ncbi.nlm.nih.gov/compound/445434#section=2D-Structure>
5. <https://pubchem.ncbi.nlm.nih.gov/compound/2153>
6. <https://pubchem.ncbi.nlm.nih.gov/compound/190>
7. Thore, S., Leibundgut, M., & Ban, N. (2006). Structure of the eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand. Science (New York, N.Y.), 312(5777), 1208–1211
8. Lorenz, R., Bernhart, S. H., Höner Zu Siederdissen, C., Tafer, H., Flamm, C., Stadler, P. F., & Hofacker, I. L. (2011). ViennaRNA Package 2.Algorithms for molecular biology: AMB, 6, 26.
9. Purzycka, K.J., Popenda, M., Szachniuk, M., Antczak, M., Lukasiak, P., Blazewicz, J., Adamiak R.W. Automated 3D RNA structure prediction using the RNAComposer method for riboswitches, Methods in Enzymology: Computational Methods for Understanding Riboswitches. (2014). 553:3-34
10. Sha Gong, Chengxin Zhang, Yang Zhang. RNA-align: quick and accurate alignment of RNA 3D structures based on size-independent TM-scoreRNA. (2019) Bioinformatics, 35: 4459-4461
11. Bocobza, S. E., & Aharoni, A. (2014). Small molecules that interact with RNA: riboswitch-based gene control and its involvement in metabolic regulation in plants and algae. The Plant journal : for cell and molecular biology, 79(4), 693–703.
12. Anthony, P. C., Perez, C. F., García-García, C., & Block, S. M. (2012). Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer. Proceedings of the National Academy of Sciences of the United States of America, 109(5), 1485–1489.
13. Mol, A. A., Vogel, M., & Suess, B. (2021). Inducible nuclear import by TetR aptamer-controlled 3' splice site selection. RNA (New York, N.Y.), 27(2), 234–241.
14. Kim, D. S., Gusti, V., Pillai, S. G., & Gaur, R. K. (2005). An artificial riboswitch for controlling pre-mRNA splicing. RNA (New York, N.Y.), 11(11), 1667–1677.
15. Ling MHT. Randomseq: python command–line random sequence generator. MOJ Proteomics Bioinform. (2018). 7(4):206–208
16. A. Krokhotin, K. Houlihan, and N. V. Dokholyan, "iFoldRNA v2: folding RNA with constraints" , 31: 2891-2893 (2015).
17. Pettersen, EF; Goddard, TD; Huang, CC; Couch, GS; Greenblatt, DM; Meng, EC; Ferrin, TE (2004). "UCSF Chimera--a visualization system for exploratory research and analysis". J Comput Chem. 25 (13): 1605–12
18. Lorenz, R., Hofacker, I.L. & Stadler, P.F. RNA folding with hard and soft constraints. Algorithms Mol Biol 11, 8 (2016).
19. S.M. Hebsgaard, P.G. Korning, N. Tolstrup, J. Engelbrecht, P. Rouze, S. Brunak: Splice site prediction in Arabidopsis thaliana DNA by combining local and global sequence information, Nucleic Acids Research, 1996, Vol. 24, No. 17, 3439-3452.
20. Arabidopsis Intron Splice-Site Tables, (2004), https://www.arabidopsis.org/info/splice\_site.pdf
21. Lukasiak, P., Antczak, M., Ratajczak, T., Bujnicki, J. M., Szachniuk, M., Adamiak, R. W., Popenda, M., & Blazewicz, J. (2013). RNAlyzer--novel approach for quality analysis of RNA structural models. Nucleic acids research, 41(12), 5978–5990.
22. Jiang, Y., & Chen, S. J. (2022). RLDOCK method for predicting RNA-small molecule binding modes. Methods (San Diego, Calif.), 197, 97–105.
23. Stefaniak, F., & Bujnicki, J. M. (2021). AnnapuRNA: A scoring function for predicting RNA-small molecule binding poses. PLoS computational biology, 17(2), e1008309.
24. Sarrion-Perdigones, A., Vazquez-Vilar, M., Palací, J., Castelijns, B., Forment, J., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2013). GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant physiology, 162(3), 1618–1631.
25. Andres, J., Blomeier, T., & Zurbriggen, M. D. (2019b, January 28). Synthetic Switches and Regulatory Circuits in Plants. Plant Physiology, 179(3), 862–884.
26. González-Grandío, E., Demirer, G. S., Ma, W., Brady, S., & Landry, M. P. (2021, September 14). A Ratiometric Dual Color Luciferase Reporter for Fast Characterization of Transcriptional Regulatory Elements in Plants. ACS Synthetic Biology, 10(10), 2763–2766.
27. Addgene: pUPD+pNOS. (n.d.). Retrieved October 2, 2022, from https://www.addgene.org/170875/
28. Sanders, P., Winter, J., Barnason, A., Rogers, S., & Fraley, R. (1987). Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants. Nucleic Acids Research, 15(4), 1543–1558.
29. CORLETT, J. E., MYATT, S. C., & THOMPSON, A. J. (1996, April). Toxicity symptoms caused by high expression of Tet represser in tomato (Lycopersicon esculentum Mill. L.) are alleviated by tetracycline. Plant, Cell and Environment, 19(4), 447–454.
30. Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., & Miyawaki, A. (2002, January). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nature Biotechnology, 20(1), 87–90.
31. Lambert, T. (n.d.). mVenus at. FPbase. Retrieved October 2, 2022, from https://www.fpbase.org/protein/mvenus/
32. Hernández, G., Osnaya, V. G., & Pérez-Martínez, X. (2019, December). Conservation and Variability of the AUG Initiation Codon Context in Eukaryotes. Trends in Biochemical Science, 44(12), 1009–1021.
33. Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2013, May 13). GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology. PLANT PHYSIOLOGY, 162(3), 1618–1631.
34. Pombo, M. A., Rosli, H. G., Fernandez-Pozo, N., & Bombarely, A. (2020). Nicotiana benthamiana, A Popular Model for Genome Evolution and Plant–Pathogen Interactions. The Tobacco Plant Genome, 231–247.
35. New England Biolabs. (n.d.). BsmBI-v2 | NEB. Retrieved October 2, 2022, from <https://international.neb.com/products/r0739-bsmbi-v2#Product%20Information>
36. New England Biolabs. (n.d.-a). BsaI | NEB. Retrieved October 2, 2022, from <https://international.neb.com/products/r0535-bsai#Product%20Information>
37. Pingoud, A., Wilson, G. G., & Wende, W. (2014, May 30). Type II restriction endonucleases—a historical perspective and more. Nucleic Acids Research, 42(12), 7489–7527.
38. Sarrion-Perdigones, A., Falconi, E. E., Zandalinas, S. I., Juárez, P., Fernández-del-Carmen, A., Granell, A., & Orzaez, D. (2011, July 7). GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLoS ONE, 6(7), e21622.
39. Goodin, M. M., Zaitlin, D., Naidu, R. A., & Lommel, S. A. (2008, August). Nicotiana benthamiana: Its History and Future as a Model for Plant–Pathogen Interactions. Molecular Plant-Microbe Interactions®, 21(8), 1015–1026.
40. Gatz, C., & Quail, P. H. (1988b, March). Tn10-encoded tet repressor can regulate an operator-containing plant promoter. Proceedings of the National Academy of Sciences, 85(5), 1394–1397.
41. Lu, J., Wu, T., Zhang, B., Liu, S., Song, W., Qiao, J., & Ruan, H. (2021b, May 22). Types of nuclear localization signals and mechanisms of protein import into the nucleus. Cell Communication and Signaling, 19(1).
42. Hebsgaard, S. (1996, September 1). Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information. Nucleic Acids Research, 24(17), 3439–3452.
43. Ho, J. M. L., Miller, C. A., Parks, S. E., Mattia, J. R., & Bennett, M. (2020b, December 8). A suppressor tRNA-mediated feedforward loop eliminates leaky gene expression in bacteria. Nucleic Acids Research, 49(5), e25–e25.
44. Berens, C., & Hillen, W. (2004b). Gene Regulation By Tetracyclines. Genetic Engineering: Principles and Methods, 255–277.
45. Lupo, A., Cesaro, E., Montano, G., Zurlo, D., Izzo, P., & Costanzo, P. (2013b, June 1). KRAB-Zinc Finger Proteins: A Repressor Family Displaying Multiple Biological Functions. Current Genomics, 14(4), 268–278.
46. Yin, J., Yang, L., Mou, L., Dong, K., Jiang, J., Xue, S., Xu, Y., Wang, X., Lu, Y., & Ye, H. (2019, October 23). A green tea–triggered genetic control system for treating diabetes in mice and monkeys. Science Translational Medicine, 11(515).
47. Sigl, R., Ploner, C., Shivalingaiah, G., Kofler, R., & Geley, S. (2014, May 19). Development of a Multipurpose GATEWAY-Based Lentiviral Tetracycline-Regulated Conditional RNAi System (GLTR). PLoS ONE, 9(5), e97764.
48. Amack, S. C., & Antunes, M. S. (2020, December). CaMV35S promoter – A plant biology and biotechnology workhorse in the era of synthetic biology. Current Plant Biology, 24, 100179.
49. Gatz, C., Frohberg, C., & Wendenburg, R. (1992b, May). Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants. The Plant Journal, 2(3), 397–404.
50. Deuschle, U., Meyer, W. K., & Thiesen, H. J. (1995b, April). Tetracycline-reversible silencing of eukaryotic promoters. Molecular and Cellular Biology, 15(4), 1907–1914.
51. Smith, A. P., Nagarajan, V. K., & Raghothama, K. G. (2011, November). Arabidopsis Pht1;5 plays an integral role in phosphate homeostasis. Plant Signaling &Amp Behavior, 6(11), 1676–1678.
52. Nagarajan, V. K., Jain, A., Poling, M. D., Lewis, A. J., Raghothama, K. G., & Smith, A. P. (2011, May 31). Arabidopsis Pht1;5 Mobilizes Phosphate between Source and Sink Organs and Influences the Interaction between Phosphate Homeostasis and Ethylene Signaling. Plant Physiology, 156(3), 1149–1163.
53. Ai, P., Sun, S., Zhao, J., Fan, X., Xin, W., Guo, Q., Yu, L., Shen, Q., Wu, P., Miller, A. J., & Xu, G. (2009, March). Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation. The Plant Journal, 57(5), 798–809.
54. Zhang, F., Wu, X. N., Zhou, H. M., Wang, D. F., Jiang, T. T., Sun, Y. F., Cao, Y., Pei, W. X., Sun, S. B., & Xu, G. H. (2014, July 25). Overexpression of rice phosphate transporter gene OsPT6 enhances phosphate uptake and accumulation in transgenic rice plants. Plant and Soil, 384(1–2), 259–270.
55. Gu, M., Chen, A., Sun, S., & Xu, G. (2016, March). Complex Regulation of Plant Phosphate Transporters and the Gap between Molecular Mechanisms and Practical Application: What Is Missing? Molecular Plant, 9(3), 396–416.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

Materials & Methods

**Materials**

##### pUPD2 (Universal GB Part Domesticator):

The pUPD2 vector is a universal domesticator plasmid for level 0 parts.
It is usually used in synthetic biology and for domestication of DNA parts for GoldenBraid assembly.
It contains a chloramphenicol resistance gene and two cloning sites (BsmBI cutting sites),
where in between there is a transcriptional unit from which β-galactosidase is expressed.

##### pDGB3α1 and pDGB3α2 vectors:

The pDGB3α vectors are universal domesticator plasmids for level α parts.
They are usually used in synthetic biology and for domestication of DNA parts for GoldenBraid assembly.
Each one contains a kanamycin resistance gene and two cloning sites (BsaI restriction sites),
where in between there is a transcriptional unit from which β-galactosidase is expressed.

##### pDGB3ω vector:

The pDGB3ω vector is a universal domesticator plasmid for level ω parts.
It is usually used in synthetic biology and for domestication of DNA parts for GoldenBraid assembly.
It contains a spectinomycin resistance gene and two cloning sites (BsmBI restriction sites),
where in between there is a transcriptional unit from which β-galactosidase is expressed.

#### 1. Bacterial strains

##### DH5α Cells (*Escherichia coli*):

DH5α Competent Cells are *Escherichia coli* cells engineered to maximize transformation efficiency.
They were used for cloning experiments.

##### GV3101 Cells (*Agrobacterium tumefaciens*):

GV3101 *Agrobacterium tumefaciens* cells were used for agroinfiltration experiments in *Nicotiana benthamiana* leaves.
This strain contains the rifampicin resistant gene (rif) and the Ti-plasmid that carry the vir genes, which are necessary for the transformation of plant cells.

#### 2. Nutrient media for bacterial cultures

##### LB Broth:

Luria Broth (LB) Broth was used in liquid form, dissolved in ddH2O and sterilized, for liquid bacterial cultures.
The desired antibiotic should also be added.

##### LB Agar:

Luria Broth (LB) Agar was dissolved in ddH2O, sterilized and added in Petri dishes for bacterial cultures.
The desired antibiotic should also be added before its solidification.

#### 3. Antibiotics

The antibiotics used were the following: ampicillin, chloramphenicol, kanamycin, rifampicin and spectinomycin.

#### 4. Enzymes

For the Restriction Enzyme Digestion and Ligation cloning experiments,
the following enzymes were used: BsaI-HF, BsmBI, Esp3I and T4 Ligase.

For the Polymerase Chain Reaction, the Q5® High-Fidelity DNA Polymerase was used.

For the diagnostic digestion experiments, the following enzymes were used:
BamHI-HF, BsaHI, BslI, EcoRI-HF, EcoRV-HF, HindIII-HF, NotI, RsaI and SspI.

#### 5. PCR Reaction

Q5 High-Fidelity 2X Master Mix was used for all the PCR experiments.

#### 6. DNA electrophoresis in agarose gel

##### TAE 50x electrophoresis buffer

The DNA electrophoresis buffer with a final volume of 1L contains 100 ml EDTA (0.5M),
242g Tris Base, 57.1 liquid acetic acid and ddH2O to final volume. The working solution is diluted from 50x to 1x.

##### Gel Loading Dye, Blue 6X

This loading solution consists of one tracking dye for agarose gel electrophoresis and contains SDS,
which often results in sharper bands.

##### NEB 1kb DNA ladder molecular weight control

##### Aqueous solution of ethidium bromide (10mg/ml)

#### 7. Reagents

Reagents for isolation of plasmid DNA from bacteria: “NucleoSpin® plasmid” kit from Macherey-Nagel

##### Ammonium molybdate

Ammonium molybdate reacts under acid conditions to form molybdophosphoric acid. In the presence of vanadium, yellow vanadomolybdo-phosphoric acid is formed, with the intensity of the yellow color being proportional to phosphate concentration. Absorbance is measured at 470 nm. Water from all sorts of samples can be used.

#### 8. Agroinfiltration Experiments

##### MM buffer

MM buffer contains 10mM MgCl2 in 10mM MES buffer. The MES buffer is a common buffering agent,
used in a lot of laboratory experiments as media, and MgCl2 is used to maintain cell homeostasis during leaf infiltration.

##### Acetosyringone

Acetosyringone is a naturally occurring phenolic compound secreted from wounded dicot plant tissues and is a chemoattractant for Agrobacterium.
It is used to attract the bacteria to the plant area and to induce the vir genes,
thus facilitating the transfer of the T-DNA region into the plant cells.

##### Hoechst 33342

Hoechst 33342 is a nucleic acid stain which emits blue fluorescence (Ex λ = 350 nm, Em λ = 461 nm) when bound to dsDNA.
It was dissolved in PBS-tween
and used for visualization of cell nuclei during the agroinfiltration samples’ observation in a confocal microscope.

#### 9. Plants

##### *Nicotiana benthamiana* WT

Leaves of the model plant *Nicotiana benthamiana* were used for the agroinfiltration experiments.

**Methods**

##### Digestion-Ligation in one pot:

The Digestion-Ligation method is based on T4 Ligase’s ability to catalyze the formation of covalent phosphodiester linkages,
which permanently attach complementary nucleotides.
The ligation reaction is used to place a desired sequence (insert) in a plasmid vector.
Usually, both of these sequences have previously been cut with the appropriate restriction enzymes,
causing the formation of sticky ends, which can be designed to be compatible with the wanted row.
T hese reactions can be catalyzed in repeated cycles to ensure a high efficiency.

|  |  |  |  |
| --- | --- | --- | --- |
||  |  |
| --- | --- |
| Reaction | 10 μL |
 Destination Vector | 50-75 ng (for pUPD2), 100-200 ng (for Alpha/Omega assemblies) |
| Part | 40-70 ng (for pUPD2), 200-400 ng (for Alpha/Omega assemblies) |
| 10X T4 DNA Ligase Buffer | 1 μL |
| T4 DNA Ligase | 1 μL |
| BsaI-HFv2 (Alpha clonings) or BsmBI-v2/Esp3I (L0 or Omega clonings) | 0.5 μL |
| ddH2O | x μL to reach 10 μL |

Use a thermocycler (PCR Machine) to run the following steps:

1. 5 min at 37℃ for BsaI/Esp3I or 42℃ for BsmBI

2. 5 min at 16℃

3. Repeat steps 1. and 2. 50-60 times

4. 10-20 min at 80℃

5. Rest at 16℃

##### Proliferation plasmid vectors from bacterial cultures

###### Competent E. coli bacteria preparation

Chemically competent cells are bacteria that have been treated with chemicals to enable them to insert exogenous DNA from their environment.
For their p reparation, the yet incompetent cells are usually treated with salts like CaCl2 and MgCl2,
whose role is the neutralization of the lipid membrane and the DNA, so as to allow the approach between them.

PROTOCOL

1. Take competent cells out of -80°C and thaw on ice

2. Mix 1-5μl of DNA into 100 μL of competent cells in a microcentrifuge or falcon tube.
GENTLY mix by flicking the bottom of the tube with your finger a few times.

3. Incubate the competent cell-DNA mixture on ice for 20-30 min.

4. Heat shock each transformation tube by placing the bottom of the tube into a 42°C heatblock for 60 sec.

5. Put the tubes back on ice for 2-5 min.

6. Add 400 μl LB (without antibiotic) to the bacteria and grow at 37°C/210 rpm for 1h.

7. Plate the transformation reaction onto a LB agar plate containing the appropriate antibiotic
and selection marker (40 μL X-gal, 10 μL IPTG for blue-white screening).

8. Incubate plates at 37°C overnight (at least 16h).

##### Verifying existence of plasmid vectors with the desired inserts

###### Small-scale isolation of plasmid DNA from bacterial cultures

To isolate and separate plasmid DNA from the rest of the cell's components and mainly from the "chromosomal" bacterial DNA,
the alkaline lysis method was used. This method is based
on the ability of plasmid DNA to rapidly reform into its double-stranded form, whereas larger DNA segments do not possess this ability.

PROTOCOL

From the bacterial colonies grown 2-3 were selected to grow overnight
in culture in the presence of liquid LB medium with the appropriate antibiotic at 37℃ with agitation.

Alkaline lysis followed, which was performed with the NucleoSpin Plasmid Mini kit for plasmid DNA
and according to the manufacturer's Isolation of high-copy plasmid DNA from E.coli protocol.
The volume of elution solution used was 40μl.

###### Diagnostic digestion with restriction enzymes

Diagnostic digestion of the plasmid DNA isolated was performed to confirm the presence of the desired insert.
Restriction endonucleases were used for this purpose.

PROTOCOL

1. Select restriction enzymes to digest your plasmid.

2. Determine an appropriate reaction buffer by reading the instructions for your enzyme.

3. In a 1.5mL tube combine the following:

|  |  |  |  |
| --- | --- | --- | --- |
||  |  |
| --- | --- |
| Reaction | 10 μL |
 DNA | 400-500 ng |
| 10x Buffer | 1 μL |
| Restriction Enzyme | 0,25 μL |
| dH2O | x μL (to bring total volume to 10µL) |

4. Mix gently by pipetting or flicking the bottom of the tube.

5. Incubate tube 37 °C for 1.5-2 hours.

###### DNA electrophoresis in agarose gel

Agarose gel electrophoresis is a technique by which DNA segments can be separated according to their molecular weight.
The substance ethidium bromide (EtBr) is used to visualize the samples during electrophoresis. When conjugated to DNA, ethidium bromide
fluoresces about 20 times more than its unconjugated form, thus making it possible to detect DNA segments in a UV table.

PROTOCOL

Sample preparation

|  |  |  |  |
| --- | --- | --- | --- |
||  |  |
| --- | --- |
| Plasmid | 1-2 μL |
 dH2O | 8-9 μL |
| Loading Dye | 2 μL |

1. Measure the appropriate volume of 1X TAE and 0,6gr of dry agarose.

2. Mix TAE and agarose in a flask and gently swirl

3. Microwave for 2-3 min (until it boils)

4. Mix it thoroughly under running water until it cools down.

5. When in the right temperature, add 2,16μl EtBr. Mix the flask to spread the EtBr everywhere.

6. Prepare the gel electrophoresis scaffold.

7. Add the liquid gel slowly into the gel box and let the gel solidify for 10-15 min.

8. Put the gel in the “electrode box”, filled with 1X TAE.

9. Load the samples in the gel wells.

10. Add the electrodes, set the gel electrophoresis instrument at 110V and run the gel for 30 min.

11. Put the gel under UV and take a photo to see what you did.

##### Integrity check of level 0 constructs

The plasmid vectors isolated were sent for sequencing to confirm the presence of the desired insert sequence.
Sequencing was done at CeMIA SA (Larissa, Greece).
The primers hybridize to the pUPD2 vector and are listed in the page PARTS of our wiki.

#### Integrity check of level 0 constructs

##### Agroinfiltration

###### Electrocompetent Agrobacterium cells preparation

Electrocompetent Agrobacterium cells are used for their ability to take up the desired plasmid via electroporation,
with the end goal of transforming plants. They are prepared by washing them with ice-cold solutions,
i.e. ddH2O and 10% glycerol solution, to remove any salts from the pellet suspension
and, therefore, to prevent any arching during their transformation and to achieve a high transformation efficiency.

PROTOCOL

Day 1:

Take from glycerol stock a small quantity and add it to the pre-culture; 5ml LB with Rifampicin 20 μg/ml

Day 3 (Evening):

1. Inoculation of a 100mL sterile LB culture with cells from the pre-culture

2. Add appropriate volume to have a final OD600 of ~ 0.03-0.04

3. Incubate at 28℃ shaking incubator for 16h

Day 4:

1. Measure the OD600 of the cells to be 0.5-0.8. If it is bigger,
dilute the culture again and re incubate it until it reaches 0.5-0.8. When you have the correct OD:

2. Place culture at 4℃ for 10-15 min to halt growth

3. Split the culture into two sterile 50 ml falcons

4. Centrifuge the falcons at 4000 x g for 10 min to harvest cells in 4℃

5. Discard the supernatant and resuspend cells with gentle swirls with ice-cold ddH2O (1X volume)

6. Repeat steps 3 and 4

7. Centrifuge the falcons at 4000 x g for 10 min at 4℃

8. Resuspend in ice-cold 10% glycerol (1/25 volume, i.e. 4 mL)

9. Centrifuge 4000 x g for 10 min and resuspend in ice-cold 10% glycerol (1/100 volume of the original culture, i.e. 1 mL or 1000 uL

10. Transfer 40 uL aliquots in 1.5 mL centrifuge tubes and snap-freeze in liquid nitrogen and store at -80℃

###### *Agrobacterium tumefaciens* Electroporation

Electroporation is a laboratory technique, used to introduce polar molecules, like DNA, to cells.
This is done by applying electrical field to cells, which leads to increased cell membrane permeability.

PROTOCOL

1. Thaw the electro-competent aliquots on ice

2. Place clean electroporation cuvettes on ice

3. Mix 1-2 μL of DNA with your thawed cells, flick gently and put back on ice

4. Transfer the DNA-cells mixture from the tube into the respective pre-chilled cuvette

5. Use the Bio-Rad MicroPulser.
Put the cuvette in the machine in the right orientation, move the cuvette towards the electrodes. Press the pulse button

6. Immediately add 900-950 μL LB in the cuvette and mix thoroughly.
After mixing, transfer the transformed cells back to the tube you used to mix cells and DNA. Incubate at 28℃/160 rpm for 2-3 hours

7. Plate cells in LB Agar plates with correct antibiotics

###### Syringe Agroinfiltration in *Nicotiana benthamiana* leaves: 28℃

The method of agroinfiltration is used to transiently transform plant leaves. *Nicotiana benthamiana*
leaves are usually preferred because of the ease of the technique, as it requires only a syringe and atmospheric pressure.

PROTOCOL

Day 1:

1. Preculture the agrobacterium strain in 5ml LB with appropriate antibiotics

2. 3 days incubation at 28℃

Day 4:

1. Inoculate 20 ml LB with 50μl of the preculture (with appropriate antibiotics)

2. 2 days incubation at 28℃ (shaking incubator)

Day 6:

1. Split the culture in 2x10ml in 50 ml falcons

2. 4.000 rpm for 10 min centrifugation

3. Remove supernatant carefully, wash the pellet with MM buffer -5ml in each falcon and resuspend GENTLY

4. 4.000 rpm for 10 min centrifugation

5. Remove supernatant and resuspend again pellet with MM buffer -5ml in each falcon

6. Gather them in 1 flask and add 200μM Acetosyringone (10μl)

7. Incubate at 28℃ at 100 rpm for 1-2h (dark)

8. Measure concentration in spectrophotometer with MM buffer as Blank. OD600 must be near 1

9. Find the correct dilution and then dilute the whole culture

10. This is now your inoculum! Put it in 2ml labeled eppendorf tubes and prepare small syringes

Infiltration

11. Turn the leaf upside down carefully and infiltrate the abaxial part of the leaf

12. First scar minimally with the syringe, then infiltrate the leaves avoiding the main veins of the leaves

13. Use a marker to circle around the infiltration points on the leaves to remember the infiltration sites and treatments

###### Observation

The working solution of Hoechst 33342 was prepared by mixing the powder into PBS-tween 1x with final concentration C = 10 ng/ml.
The solution was, then, inserted to the *N. benthamiana* leaves, via negative pressure, for the nuclei observation.
The samples were incubated for about an hour, in the dark.

After the incubation, the leaf samples were observed using confocal microscopy, with the following settings:

1. for the observation of mVenus Q69M: Ex λ = 515 nm, Em λ = 528 nm and

2. for the observation of Hoechst 33342: Ex λ = 350 nm, Em λ = 461 nm

##### Phosphorus Measurements

We used a common method for the colorimetric determination of phosphorus that is based on the reagents ammonium heptamolybdate and ammonium metavanadate (or molybdovanadate in short).
This is a very simple method. Initially, a standard curve must be calculated for the calibration, with 10 different concentrations of KH2PO4 (10mM, 9mM, 8.4mM, 6mM, 4mM, 3mM, 2.1mM, 1mM, 0.5mM and 0.1mM). Then, the phosphorus-rich water samples reactions are prepared by adding vanadate-molybdate, shaking thoroughly and waiting 10 minutes for the color to develop fully. Absorbance is measured at 470 nm.

Lab Book

It is important for every scientist to record daily all the procedures followed in the laboratory. It is practical for personal use, to be able to understand the results of experiments, and also helpful for other scientists, who might want to replicate similar experiments.
The Wet Lab members of our team understand the usefulness of such records and thus present you their Lab book.

Results

Before an attempt is made to apply our device, it is necessary to construct it, characterize it and long-term test it. In this iGEM season, we achieved to build one of the two modules that constitute the final construct of our system, modified, and partially characterize it. Moreover, we managed to build the rest of the DNA pieces needed, but their right function and characterization require more experiments.

On this page, we present you the results of the experiments conducted during this summer with the aim of the above.

**Detection Module: pNOS-TetR-(KRAB)-tNOS**

The aim of the following experiments was the construction of the transcriptional units mentioned below and their characterization in terms of their expression and intracellular localization.

We successfully built up different variations of the detection module of our system, with each one containing the Tet Repressor protein with several modifications (described in the [Design](https://2022.igem.wiki/thessaly/design) and [Engineering Success](https://2022.igem.wiki/thessaly/engineering) pages of our wiki). To be more specific, we designed the following variations:

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure1.png)

**Figure 1.** Level α variants of Detection Module.

Constructs containing the mVenus fluorescent protein were designed and built with the aim to be characterized via confocal microscopy observation, while the rest were designed and built to be used as main parts of the final Detection module.

First, the DNA fragments, ordered from IDT and TWIST, were successfully inserted to the pUPD2 vector via GoldenBraid 2.0 Cloning System (see more in our [Engineering](https://2022.igem.wiki/thessaly/engineering) page), which was confirmed by diagnostic restriction digestions (Figures 2 - 7).

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure2.png)

**Figure 2.** Diagnostic Digestion of “mVenus for TetR” with RsaI. Expected bands (bp): 1212, 796, 538 and 282. The positive results are shown by the green arrows.

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure3.png)

**Figure 3.** Diagnostic Digestion of:
(2) “KRAB” uncut,
(3) - (7) “KRAB” with EcoRI and HindIII (expected bands in bp: 2070, 417),
(8) “TetR for KRAB & Venus” uncut,
(9) – (10) “TetR for KRAB & Venus” with BsaHI (expected bands in bp: 2447, 304),
(11) “TetR for Venus, no KRAB” uncut and
(12) – (13) “TetR for Venus, no KRAB” with EcoRI and HindIII (expected bands in bp: 2070, 684)
Positive results: (4) – (7), (9), (10) and (13)
Negative results: (3) and (12)
(1) MW

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure4.png)

**Figure 4.** Diagnostic Digestion of:
(7) “pNOS for TetR” uncut,
(8) – (9) “pNOS for TetR” with EcoRI and EcoRV (expected bands in bp: 1506 and 896)
(10) “Venus cds” uncut and
(11) – (12) “Venus cds” with EcoRI and HindIII (expected bands in bp: 2070 and 756)
Positive results: (7) – (12)
(1) MW

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure5.png)

**Figure 5.** Diagnostic Digestion of:
(2), (3) “TPP riboswitch” with EcoRI and HindIII (expected bands in bp: 2473 and 308)
(4), (5) “TetR for KRAB” with BsaHI (expected bands in bp: 2446 and 307)
Positive results: (2) – (5)
(1) MW

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure6.png)

**Figure 6.** Diagnostic Digestion of “pNOS for Venus n TetR” with SspI and expected bands in bp: 1403 and 993.
(1) MW,
(2), (4), (6) and (8) digested
(3), (5) and (7) uncut
Positive results: (2), (4), (6) and (8)

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure7.png)

**Figure 7.** Diagnostic Digestion of “TetR no KRAB” with EcoRI and HindIII and expected bands in bp: 2070 and 687.
(1) MW,
(2), (4) and (6) digested
(3), (5) and (7) uncut
Positive results: (2), (4) and (6)

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure8.png)

**Figure 8.** Diagnostic Digestion of:
(2) – (4) “pNOS-TetR-tNOS” with HindIII (desired bands in bp: 6345, 964 and 513)
(5) – (7) “pNOS-Venus-tNOS” with HindIII (desired bands in bp: 6345, 1033 and 509)
(2) – (4) “pNOS-Venus-TetR-tNOS” with HindIII (desired bands in bp: 6345, 1678 and 513)
Positive results: all
(1) MW

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure9.png)

**Figure 9.** Diagnostic Digestion of:
and (3) “pNOS-Venus-TetR-KRAB-tNOS“ with HindIII (expected bands in bp: 6345, 2056 and 513)
and (5) “pNOS-TetR-KRAB-tNOS“ with HindIII (expected bands in bp: 6345, 1342 and 513)
Positive results: (2), (4) and (5)
(1) MW

Next, the level α constructs were inserted into *Nicotiana benthamiana* leaves via agroinfiltration experiments, to check their expression and their intracellular localization. The images taken during the observation of the samples are presented in Figures 10, 11 and 12.
In this experiment, ddH2O was used as negative control, the construct pDGB3α2\_pNOS-Venus-tNOS as positive control and Hoechst 33342 staining to observe the nuclei.

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure10.png)

**Figure 10.** Agroinfiltration experiment: negative (ddH2O) and positive (pDGB3α2\_pNOS-Venus-tNOS) controls.

In Figure 10, the signal of the negative control corresponds to the auto-fluorescence of the leaves and indeed it seems to appear only in circular structures that match the structure of chloroplasts. The positive control, on the other hand, emits a much stronger signal near the cell membranes. The presence of bridges, i.e. the fibers shown with red arrows, confirms that the protein is indeed being expressed in the cytoplasm and has simply been pressed towards the cell membranes due to the presence of large vacuoles, which is typical for leaf cells. It must also be mentioned that Hoechst 33342 staining efficiency was lower than expected, however for this part of our experiments it did not create significant hindrances.

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure11.png)

**Figure 11.** Agroinfiltration experiment: pNOS-Venus-TetR-tNOS.

Moving on to the main constructs of the first module, the transcriptional unit containing the Venus-TetR protein was observed in circular structures that seemed to correspond to nuclei (red arrows, Figure 11), due to their shape but also their localization within the cell, as can be seen in the merged image. In addition, the chloroplasts, visible in this image, have a much lower signal and different size, similar to those of Figure 10 (negative control). To conclude, it is safe to say that pNOS-Venus-TetR-tNOS is mainly expressed in nuclei of leaf cells.

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure12.png)

**Figure 12.** Agroinfiltration experiment: pNOS-Venus-TetR-KRAB-tNOS.

Last but not least, the construct pNOS-Venus-TetR-KRAB-tNOS was used to characterize the expression and localization of the protein Venus-TetR-KRAB. In Figure 12 a) and b), Hoechst 33342 staining efficiency was again low and thus was used to identify some nuclei where its signal was emitted more strongly. However,in both cases, where possible nuclei are observed, Venus-TetR-KRAB protein’s signal is also visible (red arrows), with higher intensity than the surrounding area. In Figure 12 c), a nucleus is present clearly, given its size and shape, the existence of which is unfortunately not confirmed by Hoechst 33342, which apparently did not work particularly well in this leaf area.

All the agroinfiltration results together, show that the TetR and TetR-KRAB proteins are expressed as expected, i.e. in the cell nucleus. Of course, more experiments like this must be conducted to confirm this conclusion.

**Nutrient Uptake Module: TetO7-pTriple-PHT1-Venus-tNOS(s)**

Aiming the characterization of the expression and intracellular localization of the second module, we successfully constructed the following transcriptional units:

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure13.png)

**Figure 13.** Level α variants of Nutrient Uptake Module.

As mentioned in the [Design](https://2022.igem.wiki/thessaly/design) and [Engineering](https://2022.igem.wiki/thessaly/engineering) pages of our wiki, we designed several variations of this module and used pht1 genes from different plant organisms with the purpose to compare their expression. All these variations would also be used as parts of the final system.

Once again, the first step was to import the DNA fragments, provided from IDT and TWIST, in pUPD2 vectors using the GoldenBraid 2.0 Cloning System (Figures 14 - 17).

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure14.png)

**Figure 14.** Diagnostic Digestion of “TetO7” with EcoRI and EcoRV and expected bands in bp: 1189, 896 and 412.
(1) MW
Positive results: (5) and (6)
Its sequence was also confirmed by sequencing.

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure15.png)

**Figure 15.** Diagnostic Digestion of “Venus for PHT1” with EcoRI and HindIII and expected bands in bp: 2070 and 759.
(1) MW,
(14) uncut and
(15) and (16) digested
Positive results: (15) and (16)

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure16.png)

**Figure 16.** Diagnostic Digestion of:
(1) “tNOS-stuffer” uncut
(2) and (3) “tNOS-stuffer” with BslI (desired bands in bp: 837, 785, 678, 279, 166 and 18)
(5) “AtPHT1;5 opt” uncut
(6) “AtPHT1;5 opt” with EcoRI (desired bands in bp: 2838 and 905)
(13) “pTriple” uncut
(14) and (15) “pTriple” with EcoRV (desired bands in bp: 1800 and 1398)
Positive results: all (“tNOS-stuffer” was confirmed by sequencing)
MW

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure17.png)

**Figure 17.** Diagnostic Digestion of:
(6) and (7) “OsPHT1;6 opt” with EcoRI and HindIII (desired bands in bp: 3387 and 332)
(10) and (11) “AtPHT1;5 non” with EcoRI (desired bands in bp: 2254, 905 and 584)
(12) “OsPHT1;6 part” with EcoRI and BamHI (desired bands in bp: 2310, 754 and 658)
Positive results: all
(1) MW

pDGB3α1\_TetO7-pTriple-Venus-tNOS(s) contained the DNA fragment “Venus cds”, that was used for the 1st module, too.

Then, cloning experiments were conducted for the construction of the level α transcriptional units, mentioned above, using the pDGB3α1 destination vector. The success of these experiments is confirmed by the following Figures 18 and 19:

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure18.png)

**Figure 18.** Diagnostic Digestion of “TetO7-pTriple-Venus-tNOS(s)” with EcoRI and HindIII and expected bands in bp: 6345, 1829, 674 and 387.
(1) MW
Positive result: (7)

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure19.png)

**Figure 19.** Diagnostic Digestion of:
(2) and (3) “TetO7-pTriple-AtPHT1;5 opt-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 1978, 1492, 674 and 387)
(4) and (5) “TetO7-pTriple-AtPHT1;5 non-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 1978, 908, 674, 584 and 387)
(6) and (7) “TetO7-pTriple-OsPHT1;6 opt-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 2041, 1405, 674 and 387)
(8) and (9) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 3449, 674 and 387)
Positive results: (2), (4) – (6) and (9)
(1) MW

The level α constructs were inserted in *Nicotiana benthamiana* leaves with the aim of observing the proteins that would be produced via confocal microscopy. Unfortunately, the confocal microscope we were supposed to use output error indication and as a consequence, we did not manage to observe the leaves and get results.

However, we decided to use the Plate Reader to obtain fluorescence emission data from our samples. To be more specific, the leaf samples from the agroinfiltration experiment mentioned above, were cut into small pieces and one leaf piece from each sample was placed at the bottom of a well. The plate was then inserted to the Plate Reader with the following settings: Ex λ = 515 nm, Em λ = 528 nm. The results of these measurements are shown in the following diagram (Figure 20):

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure20-1.png)

**Figure 20.** Agroinfiltration Experiment - Measurements of Plate Reader. GFP: leaf sample from *N. benthamiana* 16C and Negative Control: ddH2O.

From these results it appears that the positive control, i.e. pDGB3α1\_TetO7-pTriple-Venus-tNOS(s), produces a higher signal than the negative control and thus that Venus protein is expressed at high levels. Unfortunately, the measurements for the PHT1-Venus constructs are similar to the negative control, which probably means that any fluorescence observed is probably due to the autofluorescence of the leaf.

Although this procedure is not standardized in our laboratory, we can make the following conclusions: (1) the TetO7-pTriple promoter works as expected and (2) the PHT1 constructs did not expressed properly, probably due to their complex protein structure in combination with the addition of another cds (Venus).

**Final Construct**

After successfully characterizing the two modules (detection and nutrient uptake modules), we aimed to combine them into one functional device. Unfortunately, the second module did not work as expected.

However, the final constructs containing both modules in various combinations, were made (Figures 22 and 23) with the purpose of testing our whole design. The destination vector used was the pDGB3ω1 and the constructs the following:

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure21.png)

**Figure 21.** Level Ω variants of final construct.

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure22.png)

**Figure 22.** Diagnostic Digestion of:
(1) “TetO7-pTriple-Venus-tNOS(s)\_pNOS-TetR-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 2217, 1615 and 517)
(2) “TetO7-pTriple-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 2217, 1993 and 517)
(3) MW
(4) “TetO7-pTriple-AtPHT1;5 opt-Venus-tNOS(s)\_pNOS-TetR-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 3858, 1615 and 517)
(5) “TetO7-pTriple-AtPHT1;5 opt-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 3858, 1993 and 517)
(6) “TetO7-pTriple-AtPHT1;5 non-Venus-tNOS(s)\_pNOS-TetR-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 3858, 1615 and 517)
(7) “TetO7-pTriple-AtPHT1;5 non-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 3858, 1993 and 517)
(8) “TetO7-pTriple-OsPHT1;6 opt-Venus-tNOS(s)\_pNOS-TetR-tNOS” with BamHI (desired bands in bp: 6674, 3482 and 2484)
(9) “TetO7-pTriple-OsPHT1;6 opt-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” with BamHI (desired bands in bp: 6674, 3860 and 2484)
(10) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)\_pNOS-TetR-tNOS” with BamHI (desired bands in 35bp: 6674, 3482 and 2484)
(11) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” with BamHI (desired bands in bp: 6674, 3860 and 2484)
Positive results: (4) – (6) and (8)

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure23.png)

**Figure 23.** Diagnostic Digestion of:
(1) “TetO7-pTriple-Venus-tNOS(s)\_pNOS-TetR-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 2217, 1615 and 517)
(2) “TetO7-pTriple-Venus-tNOS(s)\_pNOS-TetR-tNOS” uncut
(3) “TetO7-pTriple-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 2217, 1993 and 517)
(4) “TetO7-pTriple-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” uncut
(5) “TetO7-pTriple-AtPHT1;5 opt-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 3858, 1993 and 517)
(6) “TetO7-pTriple-AtPHT1;5 opt-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” uncut
(7) MW
(8) “TetO7-pTriple-OsPHT1;6 opt-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 1993, 1793, 1350, 691 and 517)
(9) “TetO7-pTriple-OsPHT1;6 opt-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” uncut
(10) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)\_pNOS-TetR-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 2119, 1615, 964, 754 and 517)
(11) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)\_pNOS-TetR-tNOS” uncut
(12) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 2119, 1993, 964, 754 and 517)
(13) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)\_pNOS-TetR-KRAB-tNOS” uncut
Positive results: (5) and (8) – (11)

We managed to build up seven of the ten level ω constructs and, unfortunately, there was no time to test neither of them in *Nicotiana benthamiana* leaves.

**Phosphorus Measurement**

The last set of experiments that we conducted included the calibration step of the Molybdovanadate phosphate measurement method and the use of this method for the evaluation of our local river’s chemical state.

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure24.png)

**Figure 24.** Different concentrations of K2HPO4, combined with the molybdovanadate reagent during the 10 minute waiting step.

This is the standard curve that was produced from the spectrophotometer measurements:

![](https://static.igem.wiki/teams/4213/wiki/wiki/img/partnership/wetlab/results/figure25.png)

**Figure 25.** The absorbance is related to phosphorus concentration at a ratio of [P]/Abs = 7,2158.

In the end, we collected a sample from our local river and measured its absorbance (after the addition of molybdovanadate) at 0,025. This translates to a phosphorus concentration of 0,18 mM which is within the boundaries of literature data (Table 1).

|  | Phosphate levels at the Bridge of Larissa (Sample site) (mM) |
| --- | --- |
| Minimum | 0.1 |
| Maximum | 7.4 |
| Mean | 3.2 |

**Table 1.** Phosphate levels in the Pineios river. Adapted from *Mpoura N.,* (2015).

**Future Goals**

If we had successfully finished the experiments, mentioned above, and thoroughly characterized the expression of the final constructs, our next step would be the determination of their functionality. Thus, hairy root experiments, in the model plant *Lotus japonicus*, would take place, to observe the ability of the transformed roots to take up Pi at increased rates from their nutrient media.

After that, the responsiveness to microcystin-LR of the whole system would be checked. More specific, cloning experiments for the construction of variants of the Detection Module with several of the synthetic riboswitches (already designed, see more in our [Engineering](https://2022.igem.wiki/thessaly/engineering) page) and agroinfiltration experiments in absence and presence of microcystin-LR in various concentrations would be conducted.

Thus, the functional riboswitches that would stand out would be included in the final constructs, which would have to be re-characterized with the experiments analyzed above (agroinfiltration and hairy root), in absence and presence of microcystin-LR.

The variants of the final construct that would work better would continue in subsequent transformation experiments that would more closely resemble the real conditions of our project, i.e. experiments on *Phragmites australis*, nutrient media with water from Lake Karla, etc.

Of course, many of the above steps would require increased levels of biosafety measures to be applied, to ensure the safety of the environment, the society and the lab workers.

### References

1. 1. Mpoura N. (2015). Study of the burden to the Pineios river caused by nutrients. Diploma thesis at the National and Kapodistrian University of Athens

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}