iGEM 2025 — Team rubochum

{% extends "layout.html" %}
{% block title %} Notebook {% endblock %}
{% block header\_image %} https://static.igem.wiki/teams/5583/headers/notebook4x.webp {% endblock %}
{% block intro\_class %}text-only{%endblock%}

{% block intro\_text %}

## Documentation

Transparency and reproducibility are the fundamental pillars of scientific research.
To uphold these principles, we maintained a comprehensive laboratory notebook documenting every experiment, observation and insight throughout our journey.
By sharing our detailed records openly, we enable others to validate, build upon and learn from our work and therefore advancing the collective knowledge in synthetic biology.

[Notebook](https://static.igem.wiki/teams/5583/pdfs/notebook-igem-rubochum-2025.pdf)
{% endblock %}
{% block intro\_image %} {% endblock %}
{% block main\_section %}{% endblock %}
{% block page\_content %}{% endblock %}

{% extends "layout.html" %}
{% block title %} Engineering {% endblock %}
{% block header\_image %} https://static.igem.wiki/teams/5583/headers/engineering4x.webp {% endblock %}
{% block intro\_text %}

## Engineering cycle

**1. Design:** A genetic construct or method is designed based on the desired function.

**2. Build:** The designed constructs are assembled and introduced into the host organism. The method is implemented.

**3. Test:** The functionality of the constructs or method is evaluated through different experiments to verify and measure performance parameters.

**4. Learn:** Lastly the data is analysed and conclusions are drawn to improve the design for the next cycle.

{% endblock %}
{% block intro\_image %} https://static.igem.wiki/teams/5583/engineering/engcycle.webp {% endblock %}
{% block sidebar\_links %}- [Restriction Ligation cycle 1](#Restriction Ligation cycle 1)
- [Restriction Ligation cycle 2](#Restriction Ligation cycle 2)
- [Gibson assembly 1](#Gibson assembly 1)
- [Gibson assembly 2](#Gibson assembly 2)
- [Gibson assembly 3](#Gibson assembly 3)
- [DFO production & quantification](#DFO production & quantification)
{% endblock %}
{% block main\_content %}

## Restriction Ligation cycle 1

### ![](https://static.igem.wiki/teams/5583/engineering/engdesign.webp) Design

Part of our two-plasmid system contains a sevenfold repetitive sequence, referred to below as *GFP11ß*.
This repetitive structure significantly complicates both primer design and PCR implementation. Thanks to Biomatik,
the gene has already been delivered in a pET-16b plasmid with BamHI and NdeI restriction sites. The pNit plasmids we selected also have these restriction sites.
For this reason, BamHI and NdeI were chosen as restriction enzymes to integrate the *GFP11ß* gene into the pNit plasmids.

![](https://static.igem.wiki/teams/5583/engineering/engineering1-qt-rt-11.webp)

### ![](https://static.igem.wiki/teams/5583/engineering/engbuild.webp) Build

The following figures show the plasmid maps of pET16b-*GFP11ß* and pNit QT/RT.

![](https://static.igem.wiki/teams/5583/engineering/engineering2-pet16b.webp)

![](https://static.igem.wiki/teams/5583/engineering/engineering3-pnitqt1.webp)

An attempt was made to cut out the *GFP11ß* gene from the plasmid pET16b-*GFP11ß* using BamHI and NdeI restriction enzymes.
The fragment was then to be integrated into our empty pNit vector by restriction and ligation.
For digestion, both slow digestion and fast digestion were performed in NEB's CutSmart and rCutSmart buffers, respectively.
Initially, 1 µL pET16b-*GFP11ß* was used as a template.

### ![](https://static.igem.wiki/teams/5583/engineering/engtest.webp) Test

To test whether the restriction was successful, an agarose gel was run. This was done using a 1% agarose gel and run for 45 minutes at 100V.

![](https://static.igem.wiki/teams/5583/engineering/engineering4-ndfd.webp)

### ![](https://static.igem.wiki/teams/5583/engineering/englearn.webp) Learn

The restriction digestion was successful, as the product of the single digestion runs higher on the gel than that of the double digestion.
In addition, the missing DNA region corresponds approximately to the expected size of our target fragment (~700 bp).
This suggests that the *GFP11ß* fragment was successfully excised, but at a low concentration.
Since the fragment accounts for about one-tenth of the total size of the plasmid, it can be assumed that it accounts for only about 1/10 of the amount of DNA used,
i.e., approximately 100 ng.

## Restriction Ligation cycle 2

### ![](https://static.igem.wiki/teams/5583/engineering/engdesign.webp) Design

In the previous experiment, the restriction digestion was successful, but the concentration of the excised fragment was too low.
For this reason, higher starting concentrations of the plasmid were used in the further cycles to achieve a sufficient fragment yield.

### ![](https://static.igem.wiki/teams/5583/engineering/engbuild.webp) Build

For the Fast Digest, 1 µg and 5 µg of the plasmid pET16b-*GFP11ß* were used as templates. BamHI and NdeI were also used as restriction enzymes.

### ![](https://static.igem.wiki/teams/5583/engineering/engtest.webp) Test

To test whether the restriction was successful, an agarose gel was run. This was done using a 1% agarose gel and run for 45 minutes at 100V.

![](https://static.igem.wiki/teams/5583/engineering/engineering5-cycle2-test.webp)

### ![](https://static.igem.wiki/teams/5583/engineering/englearn.webp) Learn

Even an increased plasmid concentration did not lead to the desired result. In addition, the intensity of the bands in the gel decreased significantly.
Various other restriction enzymes were then tested in order to produce longer fragments and thus facilitate the isolation of the *GFP11ß* gene.
However, these attempts were also unsuccessful.

## Gibson assembly 1

### ![](https://static.igem.wiki/teams/5583/engineering/engdesign.webp) Design

After our failed attempts at creating our desired constructs using restriction/ligation, we decided to make another attempt at creating new primers to enable
amplification by PCR. The primers were created with overhangs with an approximate length of 15-20 base pairs.
These overhangs are homologous and should create sticky ends that can be used for Gibson assembly.

![](https://static.igem.wiki/teams/5583/engineering/gibson1-design1.webp)

![](https://static.igem.wiki/teams/5583/engineering/gibson1-design2.webp)

### ![](https://static.igem.wiki/teams/5583/engineering/engbuild.webp) Build

The primers were used to amplify the *GFP11ß* gene from the pET16b-*GFP11ß* plasmid. The corresponding primers were also used to amplify pNit\_QT/RT plasmids.

### ![](https://static.igem.wiki/teams/5583/engineering/engtest.webp) Test

The resulting PCR products were subsequently analyzed by running them through a 1% agarose gel for 45 min at 100 V.

![](https://static.igem.wiki/teams/5583/engineering/gibson1-test1.webp)

**Figure**
Agarosegel of Gibson Overhang PCR-products. From right to left marker (M); *GFP11ß* (gene fragment of the 11th GFP ß-strand gene fragment);
*GFP1-10* (gene fragment of the GFP ß-strands 1-10); RT (pNit\_RT1); RC (pNit\_RC1); RC1 (pNit\_RC1 from a plasmid prep as a control).

### ![](https://static.igem.wiki/teams/5583/engineering/englearn.webp) Learn

The Gel showed bands at the correct size we expected for our product which suggests that our
amplification was successful and that no unspecific binding had occurred.
We proceeded to utilize these PCR products in subsequent Gibson assembly experiments.

## Gibson assembly 2

### ![](https://static.igem.wiki/teams/5583/engineering/engdesign.webp) Design

Gibson assembly allows two fragments to be ligated together using overlapping overhangs. Three enzymes work simultaneously in the reaction.
T5 exonuclease digests the 5' end of a DNA double strand, creating sticky ends that are complementary to each other. Hybridization of the complementary
double strands occurs. DNA polymerase separates double strands and synthesizes missing pieces, and ligase connects the DNA sequences to each other, resulting
in the ligated construct. In our case, the GFP11ß gene would be inserted into the pNit-QT1/RT1 backbone.

### ![](https://static.igem.wiki/teams/5583/engineering/engbuild.webp) Build

The DNA fragments were mixed and assembled using a Hifi Master Mix that contained the necessary nucleases and ligases (see MaM for more details).
The resulting mixture was subsequently incubated at 50 °C for 60 minutes.

### ![](https://static.igem.wiki/teams/5583/engineering/engtest.webp) Test

The products of the assembly were thereafter introduced into *E.coli* Dh5α via heat shock, plated on ampicillin infused LB-Agar plates and
incubated at 37 °C overnight. No colony growth was observed.

### ![](https://static.igem.wiki/teams/5583/engineering/englearn.webp) Learn

The lack of colony growth after heatshock and incubation suggested that something went wrong during assembly, as a result of which *E. coli*
was not able to take up an intact plasmid and was subsequently eliminated by the ampicilin.

## Gibson assembly 3

### ![](https://static.igem.wiki/teams/5583/engineering/engdesign.webp) Design

Our previous attempt at Gibson assembly implied that something during the assembly process had to have gone wrong to not yield an intact construct.
Because of this we reviewed our experiment design and noticed that our overhangs of 15-18 bp were on the short end of what was described in literature.
Shorter overhangs are weaker than longer overhangs due to the fact that they are not able to produce as many hydrogen bonds holding them together.
We hypothesized that maybe the assembly temperature could be too high resulting in our overhangs perhaps overlapping but instantly separating from each other again as
a result of which no proper ligation could be performed. As a result of this we decided to perform the assembly at lower temperatures and over a longer period of time.
We chose room temperature, 30 °C and 37 °C because these temperatures were easiest to accommodate overnight with the facilities we had at our disposal.

### ![](https://static.igem.wiki/teams/5583/engineering/engbuild.webp) Build

The DNA fragments were mixed and assembled by mixing them together with a Hifi Master Mix that contained the necessary nucleases and ligases (see MaM for more details).
The resulting mixtures were subsequently incubated at 30 °C, 37 °C and room temperature overnight.

### ![](https://static.igem.wiki/teams/5583/engineering/engtest.webp) Test

The products of the assembly were thereafter introduced into *E.coli* Dh5α via heat shock, plated on ampicillin infused LB-Agar plates and incubated at 37 °C overnight.
We observed colony growth.
Colonies were picked and used to perform a colony PCR that confirmed insert integration into the MCS for 1 colony from the 30 °C assembly and 1 colonies from the 37 °C assembly.

![](https://static.igem.wiki/teams/5583/engineering/gibson3-test1.webp)

**Figure**
Agarosegel of colony PCR products of Gibson assembly heatshock colonies. Assemblies were carried out over night at 30 °C, 37 °C and room temperature (RT).
All available colonies were tested. Marker (M); Colony (C).

### ![](https://static.igem.wiki/teams/5583/engineering/englearn.webp) Learn

The results of the colony PCR suggested that assembly at lower temperatures was successful in inserting the target gene into the multiple cloning site of
the pNit\_QC Vector. Integration was subsequently also confirmed via sequencing by MicroSynth and confirmed our previous results.

![](https://static.igem.wiki/teams/5583/engineering/gibson3-learn1.webp)

**Figure**
Sequencing results alignment of pNit\_QC\_*GFP1-10* from MicroSynth with the designed sequence. The alignment shows that the insert is correctly integrated into the MCS.

## DFO production & quantification

### ![](https://static.igem.wiki/teams/5583/engineering/engdesign.webp) Design

To verify potential DFOB production, a reliable method for its extraction and quantification was required.
The design phase began with a literature review to identify a suitable approach.
This led us to the Chrome Azurol S (CAS) assay, which enables the detection of siderophores like desferrioxamine B (DFOB) in the absence of unbound iron ions.
Additionally, the CAS assay was selected to evaluate different methods for removing iron ions bound to DFOB.

### ![](https://static.igem.wiki/teams/5583/engineering/engbuild.webp) Build

DFOB was produced using *Streptomyces coelicolor* and purified with a combination of XAD-4 and XAD-16 resins.
Following elution with methanol, the DFOB was dried and resuspended in ddH₂O.
A CAS assay was then performed on the DFOB samples, alongside an EDTA standard curve,
to confirm the presence of chelators and to quantify the concentration produced.

### ![](https://static.igem.wiki/teams/5583/engineering/engtest.webp) Test

Upon extraction, the DFOB solution exhibited a brownish coloration, indicating the presence of bound iron ions.
To address this, CAS assays were conducted using DFOB samples treated with ascorbic acid, sodium carbonate, and sodium hydroxide in an effort to remove the bound iron.
The results suggested ascorbic acid, sodium carbonate and sodium hydroxide were effective in releasing iron ions.
However, similar color changes were measured in control samples containing only the additives without DFOB

### ![](https://static.igem.wiki/teams/5583/engineering/englearn.webp) Learn

The successful purification of DFOB demonstrated that the extraction method could be applied in future experiments if DFOB production in
*Rhodococcus opacus* 1CP is achieved. The CAS assay proved useful for determining DFOB concentrations. However, its sensitivity limits became apparent
when evaluating iron ion removal methods. For such applications, a more specific or less interference-prone assay should be researched.
Furthermore, removing the reducing agent or neutralizing the pH before performing the CAS assay could be beneficial.

{% endblock %}
{% block page\_content %}{% endblock %}

{% extends "layout.html" %}
{% block title %} Experiments {% endblock %}
{% block header\_image %} https://static.igem.wiki/teams/5583/headers/experiments4x.webp {% endblock %}
{% block intro\_class %}text-only{%endblock%}

{% block intro\_text %}

## Material and Methods

This section provides an overview of the experimental procedures and techniques employed during our project.
Each subsection details how a specific method was performed.

{% endblock %}
{% block intro\_image %} {% endblock %}
{% block sidebar\_links %}- [Cryo Stock preparation](#Cryo Stock preparation)
- [Primer design](#Primer design)
- [Plasmid isolation](#Plasmid isolation)
- [Polymerase chain reaction (PCR)](#Polymerase chain reaction (PCR))
- [Agarose gel electrophoresis](#Agarose gel electrophoresis)
- [Gibson assembly](#Gibson assembly)
- [Restriction digestion](#Restriction digestion)
- [Heatshock transformation](#Heatshock transformation)
- [CAS-Assay](#CAS-Assay)
- [Desferrioxamine extraction](#Desferrioxamine extraction)
- [Growth curve](#Growth curve)
- [Material](#Material)
{% endblock %}
{% block main\_content %}

## Cryo Stock preparation

For the glycerol stock, a preculture of the required organism was incubated overnight in 5 mL LB medium with the appropriate antibiotic in a test tube.
Subsequently, 1 mL of the preculture was centrifuged for 10 minutes at 6000 x g and 4 °C (Heraeus Fresco 17, Thermo Fisher Scientific Inc.). The supernatant was
discarded, and the pellet was resuspended in 500 µL of fresh medium containing the antibiotic. The culture was transferred to a sterile 1.5 mL reaction tube or a sterile
-80 °C 1.5 mL reaction tube. Approximately 500 µL of autoclaved 99% glycerol was added. The glycerol stocks were stored at -20 °C or -80 °C.

## Primer design

The primers were designed using SnapGene. All primers were designed for an annealing temperature of 60°C and provided with an overhang of 15 to 20 base pairs
to enable ligation of backbone plasmid with insert using Gibson Assembly. A cytosine or guanine was also added to the end of each primer to increase the stability
of the annealing.

## Plasmid isolation

The EasyPure® Plasmid MiniPrep Kit from TransGen Biotech was used for this purpose in accordance with the protocol provided.
The concentration of the purified plasmids was determined using the NanoDrop (NanoPhotometer NP80; Implen).

## Polymerase chain reaction (PCR)

PCR follows a 3-step structure that uses denaturation, primer annealing and elongation with the goal of exponentially amplifying specific DNA fragments.
To perform PCR a DNA template, a fitting primer pair, and depending on the provider either a master mixture or singular contents of a polymerase, free
nucleotides and a polymerase specific buffer.
The PCR samples used in this project all apply the following ratio with a total volume of 25 µl unless stated otherwise.

![](https://static.igem.wiki/teams/5583/experiments/pcr1.webp)

The following PCR method was used as a general structure and adjusted if necessary.

![](https://static.igem.wiki/teams/5583/experiments/pcr2.webp)

## Agarose gel electrophoresis

To verify the success of the PCR, the PCR products obtained were analyzed using agarose gel electrophoresis. For this purpose, a 1% agarose gel
was prepared in 1x TAE buffer (40 mM Trizma base, 20 mM acetic acid, 1 mM EDTA, pH 8.5). The gel was additionally stained with a 1:7000 dilution of
Serva DNA Stain G. For sample preparation, 10 µL of the PCR product was mixed with 1 µL of 10x FastDigest Green Buffer (Thermo Fisher Scientific, Inc.).
The GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific, Inc.) was used to determine the fragment size. Electrophoresis was performed at 100 V for 45 minutes in
1x TAE buffer. The Compact Multi-Wide System from Biometra and the Consort EV 231 power supply (Consort) were used for this purpose.
The DNA bands were detected in the UVP GelStudio System from Analytik Jena GmbH & Co.

## Gibson assembly

To combine DNA fragments that are not inherently compatible a Gibson assembly can be utilized. Following a PCR the mixture
can be subjected to a DpnI digestion to remove the sample of methylated template DNA.
Afterwards the Gibson sample can be prepared with the following structure
Samples were incubated for 1h at 50°C or at room temperature 30°C, 37°C overnight. Samples were subsequently kept on ice and stored at -20°C
for later use during heatshock transformation.

![](https://static.igem.wiki/teams/5583/experiments/gibson1.webp)

## Restriction digestion

Similar to a Gibson Assembly a restriction digestion with a follow up ligation can be utilized to introduce new genes and DNA fragments into recipient plasmids.
For this approach NdeI and Bam-HI were used as restriction enzymes, if not told otherwise.
The following sample preparation method was used for all restriction digestions unless stated otherwise.

![](https://static.igem.wiki/teams/5583/experiments/restriction1.webp)

After adding all components together, the sample is incubated at 37°C either for 1h, or 15 minutes depending on slow or fast digestion enzymes.
After the incubation the samples were loaded onto a 1% agarose gel to verify the digestion.

## Heatshock transformation

For the transformation, 2 µL of either the plasmid pET16b-11ß-fold, pBlueScript-1-10ß-fold or the cloned pNit plasmid were mixed with the cloning strain *E. coli* DH5α.
The cells were then incubated with the plasmid on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds. The cells were then placed on ice again for
10 minutes and subsequently incubated in 950 µL LB medium for 1 hour at 37°C. The cells were centrifuged for 7 minutes at 6000 x g (Heraeus Fresco 17, Thermo Fisher
Scientific Inc.), resuspended in 50 to 100 µL LB medium, and plated on agar plates with 100 µg/mL ampicillin .

## CAS-Assay

The CAS-reagent was prepared as described below. For the assay, the desired amount of sample was mixed with an equal amount of CAS-reagent.
The mixture was then incubated in the dark at room temperature for 4h. After incubation, the absorption was measured at 630 nm. This assay is based
on the paper of Alexander and Zuberer from 1991.

Alexander, D. B.; Zuberer, D. A. (1991): Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. In: Biology and Fertility of Soils 12 (1), S. 39–45. DOI: 10.1007/BF00369386.

![](https://static.igem.wiki/teams/5583/experiments/cas1.webp)

## Desferrioxamine extraction

A preculture of *Streptomyces coellicolor* was prepared in 5 ml LB medium, inoculated to an initial OD600 of 0.01 and incubated at 30°C for two days. Subsequently,
a 1 L main culture was initiated with the same starting OD600 and incubated at 30 °C for one week. After incubation, the culture was centrifuged at 4°C for 30 min
at 4000 xg. The pellet was discarded and the supernatant adjusted to pH 6.0. Then, 10 g each of XAD-4 and XAD-16 resins were added to the supernatant and the mixture
was incubated at 4 °C for 24h. The resins were removed by filtration and the bound DFOB was eluted by stepwise addition of up to 50 ml Methanol with vortexing. The
DFOB extraction based on the paper of Hofmann et al. 2021.

Hofmann, L.; Scherlach, K.; Kloss, F.; Roth, M.; Hertweck, C. (2021): Desferrioxamine E Siderophores Mediate Iron Acquisition and Virulence in the Fish Pathogen
*Streptococcus iniae*. In: Applied and Environmental Microbiology 87 (7), e02549-20. DOI: 10.1128/AEM.02549-20.

## Growth curve

Samples with a total volume of 1 mL were prepared and filled in a well plate. Each sample contained 900 µL of LB medium and was supplemented with one of the
following: 100 µL of Iron solution containing 95 nmol DFOB , 100 µl 5 mM Ascorbic acid with 95 nmol DFOB, 100 µl of 95 nmol DFOB alone, or 100 µl ddH2O (control).
The well plate was incubated at 30°C for 96 h. Measurements were taken at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h and 96 h.
After 36 h a 1:10 dilution of each sample was prepared and monitored in subsequent measurement.

## Material

In this section you can find an overview of all the materials we used during our project.

![](https://static.igem.wiki/teams/5583/experiments/medium1.webp)

![](https://static.igem.wiki/teams/5583/experiments/medium2.webp)

{% endblock %}
{% block page\_content %}{% endblock %}

{% extends "layout.html" %}
{% block title %} Results {% endblock %}
{% block header\_image %} https://static.igem.wiki/teams/5583/headers/results4x.webp {% endblock %}

{% block intro\_section %}{% endblock %}
{% block sidebar\_links %}- [Effects of desferrioxamine on *Rhodococcus growth*](#Effects of desferrioxamine on Rhodococcus growth)
- [DFO quantification via CAS-Assay](#DFO quantification via CAS-Assay)
- [Split-GFP system](#Split-GFP system)
{% endblock %}
{% block main\_content %}

## Effects of desferrioxamine on *Rhodococcus growth*

*Rhodococcus opacus* 1CP is known for its resilience and ability to grow in the presence of compounds that are toxic to many other organisms.
To determine whether desferrioxamine B (DFOB) affects the growth of *R. opacus* 1CP, we conducted growth curve experiments. These experiments
aimed to determine whether DFOB facilitates iron uptake, thereby promoting growth, or inhibits iron availability, thus restricting growth.
Growth was assessed in the presence of saturated DFOB, DFOB purified with ascorbic acid, and unpurified DFOB representing the likely state of DFOB
accumulation in an expression culture.In all cases, the presence of DFOB did not impair the growth rate of *R. opacus* 1CP (Fig. 1). However, the presence
of unpurified and saturated DFOB resulted in a shortened stationary phase, with a noticeable decline in cell density observed at the 60-hour mark. In contrast,
the control and purified DFOB samples entered the stationary phase at 48 hours and 36 hours, respectively, and maintained stable growth until the end of the
measurement period. After 60 hours of incubation, the DFO mix sample appeared to enter a decline phase, followed by a renewed growth phase at 72 hours.
This apparent reduction may be due to a measurement error, as the deviation aligns with both the preceding and subsequent measurements.

![](https://static.igem.wiki/teams/5583/results/graph1.webp)

**Figure 1**
Growth curves of *Rhodococcus opacus* 1CP in LB media (Control) under the presence of unpurified DFOB (DFO mix), with iron saturated DFOB (DFO saturated) and with ascorbic acid purified DFOB (DFOB clean).

## DFO quantification via CAS-Assay

The primary objective of our team was to introduce the necessary genes (*DesA–DesD*) into *Rhodococcus opacus* 1CP to enable more efficient production of
desferrioxamine B (DFOB). A key consideration in this context is the effect of DFOB on the growth rate of *R. opacus* 1CP. To investigate this, DFOB was
produced using *Streptomyces coelicolor* in preparation for growth curve experiments.

Several methods were tested to remove iron ions bound to DFOB. The DFOB was purified by incubating XAD-4 and XAD-16 resins in the supernatant of a one-week-old
*S. coelicolor* culture at 4 °C for 24 hours. Elution was performed by stepwise addition of methanol to the resin while rigorously vortexing.

The concentration of DFOB in the eluate was quantified using the Chrome Azurol S (CAS) assay with various dilutions. An EDTA standard curve was used for calibration.
Dilution factors were accounted for when calculating iron ion free DFOB concentrations. However, when the EDTA standard was diluted beyond a 1:10 ratio, the measured
DFOB concentrations began to decrease unexpectedly.

Subsequently, the DFOB eluate was dried and resuspended in ddH₂O. To remove iron ions bound to DFOB, another CAS assay was performed after treating the DFOB solution
with ascorbic acid, sodium carbonate, and sodium hydroxide.

All three treatments were effective in removing iron ions. However, control reactions containing only ascorbic acid, sodium carbonate, or sodium hydroxide
(without DFOB) also caused a color change in the CAS assay. This suggests that the CAS assay may not be fully reliable for assessing the efficiency of iron-ion
removal under these conditions.

![](https://static.igem.wiki/teams/5583/results/graph2.webp)

**Figure 2**
Iron ion free DFOB concentrations determined by the CAS assay using diluted elution samples. Concentrations were calculated based on an EDTA standard curve.

![](https://static.igem.wiki/teams/5583/results/graph3.webp)

**Figure 3**
Purification of DFOB assessed using the CAS assay with varying concentrations of ascorbic acid, sodium carbonate, and sodium hydroxide. Concentrations were quantified using an EDTA standard curve.

## Split-GFP system

To monitor gene cluster activity during the transfer of the desferrioxamine B (DFOB) biosynthesis genes (*DesA–DesD*) into *Rhodococcus opacus 1CP*, we aimed to implement a split-GFP system.
For our split GFP system to be compatible with *Rhodococcus opacus 1CP*, we first needed to transfer the relevant genes into pNit vector systems. We successfully cloned
the *GFP1-10* fragment into the pNit\_QC1 vector and the *GFP11ß* into the pNit\_QT1 resulting in the plasmids pNit\_QC1\_*GFP1-10* and pNit\_QT1\_*GFP11ß*. Both plasmids were verified via sequencing.
Unfortunately we were not able to test the split GFP system in *Rhodococcus opacus 1CP* due to time constraints. Testing in *E.coli* was also not feasible since
pNit\_QT1 and pNit\_QC1 cannot simultaneously be transformed into *E. coli* since they share the same origin of replication and ampicillin resistance.

{% endblock %}
{% block page\_content %}{% endblock %}