iGEM 2025 — Team stuttgart

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# Our engineering success

**Design.** Every project starts with an idea. This idea is then refined into a concise plan.

**Build.** The plan is then applied. DNA constructs are assembled.

**Design.** The design is put to the test. Does it work? How does it perform?

**Learn.** The takeaways from this cycle are gathered. Improvements are made. The cycle begins again.

×
![]()

## The biological cycles

Cycle 1

Cycle 2

Cycle 3

Cycle 4

## Cycle 1 – testing *pEKEx2* & *pPBEx2* as expression systems

## Design

Our team successfully engineered the bacterium *Corynebacterium glutamicum* to produce oxalic acid, a valuable chemical used
in everything from cleaning products to pharmaceuticals. We used *C. glutamicum* because it’s a proven industrial workhorse,
and we gave it a new ability by borrowing a gene from the fungus *Aspergillus niger* [[Ray et al, 2022](#Ray)].

The key was the oxaloacetate hydrolase (*OAH*) gene. In theory, implementing *OAH* in C. glutamicum’s metabolism should lead to the conversion of oxaloacetate to oxalate and acetate:

![Description](https://static.igem.wiki/teams/5829/images/metabolicpathway.webp)

For optimal translation conditions, we chose a codon-optimized variant of *oahA*. Additionally, we added a His6Tag a the c-terminus so that *OAH*-purification is possible.
As expression systems, we decided to test the IPTG-inducible expression vector *pEKEx2*, which is well-established at our institution as well as ist derivative *pPBEx2*.
The latter was designed by [Bakkes et al.](#Bakkes) (Jülich, Germany) and should present with lower leakiness.

## Build

Codon optimized *oahA* was ordered from Gigabases, a DNA synthesis company from Switzerland. The gene of interest was subcloned into linearized *pEKEx2*
and *pPBEx2* by amplifying it via Q5-PCR and using the NEBuilder DNA Assembly protocol for assembly. After successfully creating *pEKEx2\_oahA* and *pPBEx2\_oahA*,
both vectors were transformed into *E. coli* and subsequently electroporated into the target organism *C. glutamicum*.

## Test

To test the production capability of our new mutants as well as their viability, we conducted a first cultivation with both mutants and the wildtype as control.
Oxalate production was induced by adding 2.5 mM IPTG after 3 h of cultivation. Two mutant cultures were not induced to test for leakiness.
The extracellular oxalate level was measured after 6 hours using a commercially available enzymatic assay and biomass formation was tracked by measuring optical density (OD600).

The results show that our mutants indeed produce oxalic acid and their growth rate is only slightly decreased in comparison to the wildtype.

![First growth curve](https://static.igem.wiki/teams/5829/images/growthcurve1.webp)

Growth rates in exponential phase. Induced mutants show slightly decreased values.

| culture | µmax |
| --- | --- |
| WT | 0.43 |
| *pEKEx2* | 0.42 |
| *pEKEx2*+ | 0.37 |
| *pPBEx2* | 0.40 |
| *pPBEx2* | 0.38 |

![First growth curve](https://static.igem.wiki/teams/5829/images/oxalate1.webp)

| culture | Oxalate[µM] |
| --- | --- |
| *C.g.* Wildtype | 74 |
| *C.g. pPBEx2\_oahA* induced | 360 |

We also measured oxalate concentration kinetics throughout the whole cultivation (data not shown), however
since the results showed extremely high variance they were not reliable.

To quantify oxalate production and further evaluate growth characteristics in larger scale, we tested *C.g. pPBEx2\_oahA*
in a 1 L stirred tank reactor system. The results validated our previous data and confirms that the growth rate is
decreased by introducing the *pPBEx2\_oahA* plasmid.

![Justice scale](https://static.igem.wiki/teams/5829/images/growthcurve-largescale.webp)

Oxalate production was again tested using the same enzymatic kit as before. The results once again showed extremely high variance.
The only take-away is that the plasmid-containing induced cultures showed elevated oxalate levels while the wildtype cultures only showed traces.

## Learn

This first cycle provided us with a proof of concept. We learned that the plasmid burden on the organism is reasonable and
oxalate production is possible with our approach. The unreliable oxalate data remain a problem which we will try to solve by
testing different quantification methods including HPLC and a chemical precipitation assay (see cycle:
[Oxalate Assay](contribution.html)).

To further optimize oxalate production, we decided to try genome integration of the *oahA* gene for a more stable production without
having to rely on antibiotics. Apart from that, different induction times and inducer concentrations should be tested to find the optimal conditions.

‹

›

## Cycle 2 – genome integration trial 1 – integration of *oahA*

## Design

For the first genome integration trial, we chose to integrate the *oahA* gene under a *tac* promoter via 2 step homologous recombination
by introducing an integration plasmid containing our insert. Upon the recommendation of our experts, we used *pK19mobsacB* as the integration vector.
After extensive literature research, we identified the region between *cg3228* and *cgIldA* as a suitable integration locus [[Lange et al., 2017](#lange)].

![first integration](https://static.igem.wiki/teams/5829/images/firstintegration.webp)

The first goal is to create *pK19\_oahA* (*pK19* containing *oahA* with *Ptac* and *lac* operator derived from *pEKEx2\_oahA* as well as
two adapter regions for homologue recombination) and transforming it into *E. coli* for plasmid amplification.
After that, we can electroporate the isolated plasmid into our target organism *C. glutamicum* for genome integration.

## Build

The integration vector *pK19\_oahA* was built by first creating fragments of (1) the upstream adapter region,
(2) the *oahA* fragment containing *Ptac* and *lac* operator and (3) the downstream adapter region via Q5-PCR.
The fragments were amplified with overlapping primers so that DNA assembly using NEB’s assembly protocol was possible.
The 3 fragments were assembled into the BamHI/EcoRI-linearized *pK19* vector.

## Test

Correct assembly was tested by (1) gel electrophoresis of the assembly reaction and (2) transforming the plasmid into
*E. coli* followed by colony PCR, gel electrophoresis and sequencing. Gel electrophoresis of the assembly reaction showed promising results,
however after checking the grown colonies with sequencing, no positive clones could be confirmed:

![sequencing](https://static.igem.wiki/teams/5829/images/sequencing-firstintegration.webp)

Sequencing results aligned with the expected sequence. Each line shows one tested sequence. Red indicates matches, blank indicates mismatches/missing basepairs.

## Learn

The sequencing results all have in common that no tested sequence contains a functional *tac* promoter. This lead us to conclude
that the plasmid is too high of a burden for *E. coli* if not regulated since the expressed *oahA* gene constantly removes oxaloacetate from the TCA cycle.
This means that no cell containing the whole *Ptac-oahA* sequence could survive which is exactly what we see in the sequencing results.
To solve this problem, we decided to try integrating an additional repressor system alongside the oahA and Ptac to inhibit constant OAH-expression.

‹

›

## Cycle 3 – genome integration trial 2 – integration of *oahA* and *lacI*

## Design

To lighten the burden of *pK19\_oahA*, we decided to integrate the *lac*-repressor system:

![first integration](https://static.igem.wiki/teams/5829/images/secondintegration.webp)

Without addition of the inducer IPTG, the repressor system consisting of lacI, its promoter *lacIq* and the *lac* operator
should inhibit the expression of *oahA*. LacI is a DNA binding protein which binds to the *lac* operator and inhibits transcription
of subsequent genes [[Bell, Lewis, 2001](#bell)].

The first step to genome integration is once again building the integration vector, this time with the lacI system: *pK19\_lacI\_oahA*.

## Build

*pK19\_lacI\_oahA* was created by constructing 4 overlapping fragments via Q5-PCR: (1) Upstream adapter region,
(2) *lacIq*+lacI, (3) *Ptac-lac* operator-oahA, (4) Downstream adapter region. These fragments were assembled into
linearized *pK19* following NEB’s DNA assembly protocol.

## Test

After assembly, the reactions were directly transformed into *E. coli* by heat shock. After selection on agar plates containing Kanamycin overnight,
the surviving colonies were checked with colony PCR and subsequent gel electrophoresis. Unfortunately, no positive colonies had formed:

![Justice scale](https://static.igem.wiki/teams/5829/images/gel-thirdintegration.webp)

Expected size of the correctly assembled fragment was 4170 bp. However, none of the 40 tested colonies contained the right fragment; most had a size of 500-1000 bp.

## Learn

The results proved our previous theory wrong; integrating a repressor system alone does not lead to survival of correctly transformed colonies.
We suspected that one cause could be that we did not add a terminator to our *oahA* gene.
To eliminate this problem we decided to try including the T7 terminator and see how this influences colony formation.

‹

›

## Cycle 4 – genome integration trial 3 – integration of *oahA* with lacI and terminator

## Design

To design a functional integration vector, we decided to add the [T7 terminator](https://parts.igem.org/Part%3ABBa_B0012) to our *oahA* unit:

![fourth integration](https://static.igem.wiki/teams/5829/images/fourthintegration.webp)

The terminator is supposed to end transcription after reaching the end of the protein coding sequence *oahA* which should decrease energy demand.

## Build

This time, the whole fragment was ordered from TwistBiosciences to eliminate any assembly mistakes. The fragment was amplified using Q5-PCR
and cloned into the linearized *pK19* vector. This time, *pK19* was linearized with HindIII and BamHI.

## Test

The assembled *pK19\_lacI\_oahA\_TE* was tested by transforming it into *E. coli* and testing for positive colonies with cPCR after selection on Kan50 agar plates.
However, the gel electrophoresis of the PCR-products showed no positive results:

![gel](https://static.igem.wiki/teams/5829/images/gel-fourthintegration.webp)

Expected length was 3642 bp -> no positive clones

## Learn

We learned that adding a terminator alongside the *lac*-repressor system did not help with decreasing the metabolic burden on *E. coli*.
After some further literature research, we found that a possible problem could be the high copy number of *pK19* (~500).
In comparison, the expression vector *pEKEx2* we used in previous experiments only has a copy number of 30.
Possible solutions could be chossing a different integration vector with medium copy number for *E. coli* or directly electroporating
the assembly product into *C. glutamicum* for direct genome integration. Although not optimal due to low plasmid concentrations,
this would eliminate the need for a plasmid that is suitable not only for *C. glutamicum* but also for *E. coli*.
This will be tested in further experiments.

‹

›

---

## Chemistry

## Design

Oxalate ions can form soluble metal oxalato complexes with PGMs like platinum, predominately as [Pt(C₂O₄)₂]²⁻
[[Krishnamurty, 1961](#Krishnamurty)].

To make our own claims about PGM yields from leaching with oxalic acid, we designed a simple experimental set-up
with varying parameters to determine the optimal conditions. For this, we used spent catalysts ground-up into
PGM dust (courtesy of Mairec) that was mixed with oxalic acid and heated up over an oil bath. The resulting solution
with the metal oxalato complexes was filtered and distilled. The samples were analyzed via inductively coupled plasma
optical emission spectroscopy (ICP-OES).

## Build

The experiment was set up as follows: The PGM dust and the oxalic acid were deposited into a
three-neck round-bottom flask placed in an oil bath and solved in water. The leaching process
takes several hours, during which the oxalate ions form soluble complexes with the PGMs.

![Justice scale](https://static.igem.wiki/teams/5829/images/chemprocess.webp)

![Justice scale](https://static.igem.wiki/teams/5829/images/chemprocess2.webp)

Then, the solution is filtered to separate the complexes solved in the solution from the remaining
unsolved PGM dust components. This time is also used to heat up the oil bath to >100°C to evaporate
the water, leaving behind the desired metal oxalato complexes. This is done with the help of a reflux
cooler. The dry sample was then analyzed via ICP-OES.

## Test

The ICP-OES analysis offered the following results.

![First growth curve](https://static.igem.wiki/teams/5829/images/pgmmeasurement.webp)

PGM measurement of the three samples and reference via ICP-OES in mg⁄L

In this simple set-up, it seems that no other PGMs than platinum were leached by the oxalic acid.
The leaching rate for Pt is relatively low compared to rates acquired from leaching cycles performed
with *A. niger* [37% [Malekian, 2019](#Malekian)].

## Learn

These first rudimentary cycles confirmed that we could leach platinum out of spent catalysts with oxalic acid.
Yet, it also proved that both the analysis of our samples as well as the complexity of the experimental set-up
to be more complicated than previously anticipated.

We drew the following conclusions:

* We needed to reach out to chemists in the field who could
  provide necessary know-how on how and where we could analyze our samples.
* To improve the leaching yield, we should pretreat the
  PGM dust. This started a lengthy process of literature research, which helped us finalize the process in theory.

‹

›

## References

1. Ray, D. et al. (2022). The soil bacterium, Corynebacterium glutamicum, from biosynthesis of value-added products to bioremediation: A master of many trades. Environmental Research, 213, 113622. URL: <https://doi.org/10.1016/j.envres.2022.113622>

2. Bakkes, P. J. et al. (2020). Improved pEKEx2-derived expression vectors for tightly controlled production of recombinant proteins in Corynebacterium glutamicum. Plasmid, 112, 102540. URL: <https://doi.org/10.1016/j.plasmid.2020.102540>

3. Lange, J. et al. (2018). Harnessing novel chromosomal integration loci to utilize an organosolv-derived hemicellulose fraction for isobutanol production with engineered Corynebacterium glutamicum. Microb Biotechnol., 11(1), 257-263. URL: <https://doi.org/10.1111/1751-7915.12879>

4. Bell, L. (2001). The Lac repressor: a second generation of structural and functional studies. Current Opinion in Structural Biology, 11, 1, 19-25. URL: [https://doi.org/10.1016/S0959-440X(00)00180-9](https://doi.org/10.1016/S0959-440X%2800%2900180-9)

5. Krishnamurty, K. V. et al. (1961). The Chemistry of the Metal Oxalato Complexes. Chem. Rev., 61, 213-246. URL: <https://doi.org/10.1021/cr60211a001>

6. Malekian, H. et al. (2019). Investigation of platinum recovery from a spent refinery catalyst with a hybrid of oxalic acid produced by Aspergillus niger and mineral acids. Waste Manag, 85, 264–271. URL: <https://doi.org/10.1016/j.wasman.2018.12.045>

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## Contents

[Proof of concept](#proofofconcept)
[Scale-up DASGIP](#dasgip)
[Optimization - cultivation parameters](#parameters)
[Optimization - genome integration](#integration)
[Oxalate analysis](#oxalate)
[Leaching results](#chem)

# Results

Our objective regarding biotechnology was to engineer an oxalic acid producer that is

* Safe
* Easy to handle and
* Produces high quantities of oxalic acid for bioleaching of PGMs.

×
![]()

## Proof of concept

---

## Creating an oxalate producer

The first big goal was to genetically engineer *Corynebacterium glutamicum* to produce oxalic acid. In order to achieve this,
we introduced the codon-optimized *oahA*-gene originating from *Aspergillus niger* into the expression vectors *pEKEx2* and *pPBEx2* by DNA assembly using NEB’s assembly protocol.
*OahA* encodes the oxaloacetate acetylhydrolase which converts oxaloacetate into oxlaate and acetate. Since oxaloacetate is a key metabolite in the TCA cycle,
it was important to check whether the cells could even survive this interference.

The two plasmids were transformed into *E. coli* and subsequently electroporated into *C. glutamicum*. Colony PCR followed by gel electrophoresis confirmed the successful transformation (Fig. 1).

![First growth curve](https://static.igem.wiki/teams/5829/images/proofofconcept.webp)

The positive clones were sent for sequencing which confirmed the positive gel electrophoresis results.

---

## First cultivation experiment

Viability and production-capability of the plasmid-containing mutants *C. glutamicum pEKEx2\_oahA* and *C. g. pPBEx2\_oahA* were tested in a first cultivation:

Five 50 mL cultures on CGXII-medium in baffled shake flasks were inoculated with

* *C.g.* wildtype
* *C.g. pEKEx2\_oahA*
* *C.g. pEKEx2\_oahA* (+)
* *C.g. pPBEx2\_oahA*
* *C.g. pPBEx2\_oahA* (+)

and cultivated until growth stagnated. Cultures 3) and 5) were induced with 2 mM IPTG after 3 hours to start oxalate production.
Optical density was measured every 1.5 hours and oxalate levels were quantified using a commercial enzymatic oxalate assay kit.

Results showed that the growth rate of plasmid containing cultures was only slightly decreased in comparison to the wildtype.
This prooves viablity and good growth performance on CGXII-medium.

![First growth curve](https://static.igem.wiki/teams/5829/images/growthcurve1.webp)

Table 1: Growth rates in exponential phase. Induced mutants show slightly decreased values.

| culture | µmax |
| --- | --- |
| WT | 0.43 |
| *pEKEx2* | 0.42 |
| *pEKEx2+* | 0.37 |
| *pPBEx2* | 0.40 |
| *pPBEx2* | 0.38 |

As expected, growth rates of induced cultures are lower than those not induced. This is a first hint that oxalate production was
indeed induced upon addition of IPTG which increases the metabolic burden. Oxalate quantification unfortunately showed no reliable results.

![First growth curve](https://static.igem.wiki/teams/5829/images/oxalatelevels.webp)

Fig. 3 shows that oxalate was produced in the induced cultures but not/only to a low extend in the uninduced controls as well as the wildtype.
However, oxalate levels after 28 h in the induced cultures show extremely high variance (156235.3 µM vs. 1239.0 µM for *pEKEx2\_oahA* and *pPBEx2\_oahA*, resprectively).
This can only be attributed to errors in measurement, since it is highly unlikely that two plasmids so similar behave so differently at the end of cultivation.
In conclusion, oxalate production can be proved qualitatively but no quantitative results are obtained at this point.

---

## Conclusion

The key findings for this first stage are:

* *C. g. pEKEx2\_oahA* and *pPBEx2\_oahA* are not only viable but show excellent growth performance
* Oxalate production is possible by introducing just one new enzyme in *C. glutamicum’s* metabolism
* Oxalate is exported out of the cells
* Oxalate quantification proves difficult

Open questions are:

* How much oxalate can be produced?
* How much oxalate remains intracellular?
* How can oxalate production be increased?

---

## Scale-up DASGIP

![First growth curve](https://static.igem.wiki/teams/5829/images/dasgipreactor.webp)

DASGIP bioreactors are stirred-tank reactors with a working volume of 1.5 liters. All reactors were equipped with Rushton turbines.
In this experiment, *Corynebacterium glutamicum*, a model organism commonly employed in bioprocess engineering, was cultivated in six parallel reactors
with CGXII medium to examine the impact of oxalate production on bacterial growth kinetics. The wild type was cultivated in three reactors,
while the remaining three reactors contained the bacterium with the plasmid *pPBEx2\_oahA* for oxalate production.
This plasmid contains a repressor system in which the gene for oxalate production is only transcribed upon addition of IPTG.
After 3 hours of cultivation, two *pPBEx2\_oahA* cultures were induced with 2 mM IPTG to investigate the effects of oxalate production on growth,
while one wild-type culture was induced with IPTG as a control to assess the effects of the inducer itself on growth.
Samples were taken hourly throughout the experiment to determine the OD₆₀₀, biomass dry weight, and oxalate concentration.
The experiment was terminated when exhaust gas analysis indicated an increase in O₂ content in the off-gas, signifying that the bacteria no longer
consumed the supplied oxygen. This occurred after approximately 10 hours for the wild type and after approximately 14 hours for the plasmid-bearing cultures.

## Kinetic and yield parameters of the reactors

To quantitatively characterize and compare the growth behavior of the different cultures, the maximum growth rate (µmax),
the biomass yield on substrate (glucose) (YX⁄Glc), and the specific glucose uptake rate (qGlc) were calculated from the measurement data
and are given in the following table.

![First growth curve](https://static.igem.wiki/teams/5829/images/tabelle-mit-einheiten.webp)

## Maximum growth rate (µmax)

The wild type achieved a maximum specific growth rate of 0.41 h-1. The non-induced plasmid-bearing culture exhibited a reduced growth rate of 0.33 h-1,
representing a 19.51% decrease. The IPTG-induced plasmid cultures displayed a further reduction of 4.88%,
corresponding to a maximum specific growth rate of 0.31 h-1 and thus a total reduction of 24.39% compared to the wild type.

This reduction can be attributed to the increased metabolic burden imposed by the plasmid. The bacteria must allocate additional
energy and resources for plasmid replication and expression of the kanamycin resistance gene encoded on the plasmid, as well as,
following IPTG addition, for the expression of the *oahA* gene responsible for oxalate production.

The IPTG-induced wild-type culture exhibited a growth rate of 0.40 h-1, comparable to that of the non-induced wild type.
This indicates that IPTG itself does not exert a significant influence on the growth of *C. glutamicum*.

## Biomass yield per glucose (YX⁄Glc)

During the determination of the biomass yield on glucose, relatively high standard deviations were observed for both the wild type and the plasmid-bearing strain.
To assess whether the mean values differ significantly, a Student's t-test was performed. The resulting p-value did not indicate significance,
suggesting that the biomass yield on glucose does not differ between the strains. This outcome contrasts with our initial hypothesis that the yield
would decrease in the plasmid-bearing strains, as part of the substrate is redirected towards oxalate production.
The reason for this is presumably that the amount of oxalate produced by the bacteria is too low to cause a measurable change in biomass substrate yield.

## Specific glucose uptake rate (qGlc)

The specific glucose uptake rate was 0.19 g⁄(g·h) for the wild type and 0.14 g⁄(g·h) for the plasmid-bearing cultures,
representing a 19% reduction that corresponds to the decreased maximum specific growth rate.

## Oxalate Production

To measure oxalate production in samples from Reactor 1 and Reactor 3, an enzymatic oxalate assay kit from Sigma-Aldrich (MAK315) was used.
This assay employs HRP and OX enzymes to catalyze a colorimetric reaction, with color intensity being directly proportional to oxalate concentration.
The optical density values of samples from Reactor 1 (Fig. 4) demonstrate qualitatively that bacteria harboring the induced *pEPEX2\_oahA* plasmid exhibited
increasing oxalate production over the course of cultivation. No oxalate production was detected in the wild-type strain.

![Scale-up growth curve](https://static.igem.wiki/teams/5829/images/oxalatproduktion.webp)

Figure 4: Optical density values of samples from Reactor 1 show oxalate production by the *pEPEX2\_oahA* containing bacteria.

Due to an imprecise standard curve and the limited number of samples that could be analyzed with this assay, duplicates or triplicates were not possible,
and the quantitative results lack validity. Only a rough estimate can be provided: approximately 582.8 µM oxalate was produced after 15 hours.

Due to the high costs, limited sample numbers, and unsatisfactory results in the evaluation of this and previous cultivation experiments,
the development of an in-house oxalate assay was initiated concurrently.

## Conclusion

The results of this bioreactor cultivation experiment provide clear evidence for the metabolic cost of oxalate production in *C. glutamicum*.
Plasmid-bearing strains showed a 19.51% reduction in maximum specific growth rate and a 19% decrease in specific glucose uptake rate compared to the wild type.
Notably, IPTG induction further reduced the growth rate by 4.88%, indicating that active oxalate production imposes an additional burden beyond plasmid maintenance.
The cell dry weight over time, depicted in the following figure, visually confirms this growth impairment, with plasmid-bearing cultures requiring
approximately 4 additional hours to reach stationary phase. The absence of a significant change in biomass yield on glucose suggests that the amount of oxalate secreted by the bacterium was too low to exert a measurable effect.
These findings provide a quantitative baseline while revealing substantial optimization potential for future strain engineering to enhance oxalate production.

![Scale-up growth curve](https://static.igem.wiki/teams/5829/images/growthcurve-largescale.webp)

Figure 5: The growth curve of the DASGIP experiment shows decreased growth for oxalate producing variants.

---

## Optimization

---

## Cultivation parameters

To elevate oxalate production, different induction times and inducer concentrations were tested to find the optimal conditions.
However, results were inconclusive (data not shown) and need to be repeated in future experiments.

---

## Genome integration

Another approach to enhance oxalate production is to create a more stable production strain by integrating *oahA* into the genome.
This way, no more antibiotics are needed for prevention of plasmid-loss. If integration is successful, the next step is to introduce
the plasmid again to see how the combination of plasmid and genome integrated *oahA* influences the production.

For genome integation, we chose *pK19mobsacB* as the integration vector. *pK19* contains an origin of replication for *E. coli*,
which means that it can be amplified in *E. coli* for subsequent integration in *C. glutamicum‘s* genome via 2 step homologue recombination.
After extensive literature research, we identified the region between *cg3228* and *IldA* as a suitable locus [[Lange et al., 2017](#lange)].

Three different attempts were carried out:

* Integration of *oahA*

  ![First growth curve](https://static.igem.wiki/teams/5829/images/intoaha.webp)

  Figure 6: Integration of *oahA* into *cg3228/IldA*.
* Integration of *oahA* with addition of a *lacI* repressor system
  ![First growth curve](https://static.igem.wiki/teams/5829/images/secondintegration.webp)

  Figure 7: Integration of *oahA* with *lacI* repressor system into *cg3228/IldA*.
* Integration of *oahA* with *lacI* repressor system and terminator
  ![First growth curve](https://static.igem.wiki/teams/5829/images/fourthintegration.webp)

  Figure 8: Integration of *oahA* with *lacI* repressor system and terminator into *cg3228/IldA*

* Integration of *oahA*

  In the first trial, *oahA* with *tac* promoter and *lac* operator was assembled into *pK19* together with Upstream and
  Downstream adapters homologue to the *C. glutamicum* genome integration locus flanks.

  ![First growth curve](https://static.igem.wiki/teams/5829/images/pk19oaha.webp)

  Figure 9: Integration vector *pK19\_oahA* containing *oahA* under *Ptac* and with *lac* operator. Upstream and Downstream flanking regions are homologue
  to the integration locus in the *C. g.* genome. *pK19* contains *SacB* and *KanR* for two-step selection.

  After assembly, *pK19\_oahA* was introduced into *E. coli* via heat shock. After selection on Kan50 agar plates, colonies were tested with
  cPCR for correctly assembled plasmids and sent for sequencing. However, no positive clones could be confirmed.

  ![First growth curve](https://static.igem.wiki/teams/5829/images/sequencing-firstintegration.webp)

  Figure 10: Sequencing results aligned with the expected sequence. Each line shows one tested sequence. Red indicates matches, blank indicates mismatches/missing basepairs.

  The sequencing results all have in common that no tested sequence contains a functional tac promoter. This led us to conclude that
  the plasmid is too high of a burden for *E. coli* if not regulated since the expressed *oahA* gene constantly removes oxaloacetate from the TCA cycle.
  This means that no cell containing the whole *Ptac-oahA* sequence could survive which is exactly what we see in the sequencing results.
* Integration of *oahA* with addition of a *lacI* repressor system

  To solve this problem, we decided to try integrating an additional *lacI*-repressor system alongside the *oahA* and *Ptac* to inhibit constant *OAH*-expression.
  Without addition of the inducer IPTG, the repressor system consisting of *lacI*, its promoter *lacIq* and the *lac* operator should inhibit the expression of *oahA*.
  LacI is a DNA binding protein which binds to the *lac* operator and inhibits transcription of subsequent genes [[Bell, Lewis, 2001](#bell)].

  After assembly, the reactions were directly transformed into *E. coli* by heat shock. After selection on agar plates containing Kanamycin overnight,
  the surviving colonies were checked with colony PCR and subsequent gel electrophoresis. Unfortunately, no positive colonies had formed:

  ![First growth curve](https://static.igem.wiki/teams/5829/images/gel-thirdintegration.webp)

  Figure 11: cPCR results. Expected size of the correctly assembled fragment was 4170 bp. However none of the 40 tested colonies contained the right fragment; most had a size of 500-1000 bp.

  The results proved our previous theory wrong; integrating a repressor system alone does not lead to survival of correctly transformed colonies.
  We suspected that one cause could be that we did not add a terminator to our *oahA* gene. To eliminate this problem we decided to try including the T7 terminator and see how this influences colony formation.
* Integration of *oahA* with *lacI* repressor system and terminator

  To design a functional integration vector, we decided to add the [T7 terminator](https://parts.igem.org/Part%3ABBa_B0012)
  to our oahA unit. The terminator is supposed to end
  transcription after reaching the end of the protein coding sequence *oahA* which should decrease energy demand.

  This time, the whole fragment was ordered from TwistBiosciences to eliminate any assembly mistakes. The fragment was amplified
  using Q5-PCR and cloned into the linearized *pK19* vector. This time, *pK19* was linearized with HindIII and BamHI.

  The assembled *pK19\_lacI\_oahA\_TE* was tested by transforming it into *E. coli* and testing for positive colonies with cPCR after selection on Kan50 agar plates.
  However, the gel electrophoresis of the PCR-products showed no positive results.

  ![First growth curve](https://static.igem.wiki/teams/5829/images/gel-fourthintegration.webp)

  Figure 12: Results of *E. coli pK19\_lacI-oahA\_TE* cPCR. Expected length was 3642 bp -> no positive clones.

  We learned that adding a terminator alongside the lac-repressor system did not help with decreasing the metabolic burden on *E. coli*.
  After some further literature research, we found that a possible problem could be the high copy number of *pK19* (~500).
  In comparison, the expression vector *pEKEx2* we used in previous experiments only has a copy number of 30.
  Possible solutions could be choosing a different integration vector with medium copy number for *E. coli* or directly electroporating
  the assembly product into *C. glutamicum* for direct genome integration. Although not optimal due to low plasmid concentrations,
  this would eliminate the need for a plasmid that is suitable not only for *C. glutamicum* but also for *E. coli*.
  This will be tested in further experiments.

---

## Conclusion

The failed genome integration remains to be solved. Possible solutions are:

* Using a different integration vector with lower copy number
* Directly electroporating the integration vector in *C. glutamicum*

Cultivation parameters need to be further tested to increase oxalate production. Apart from inducer concentration and induction time,
we want to test different supplements (e.g. ethanol) and their influence on production.

Our main problem however is oxalate quantification. With the commercial oxalate assay kit, no reliable results could be obtained.
To address this issue, we tried measuring oxalate using HPLC and even designed our own oxalate assay with a precipitation based approach.

---

## Oxalate measurements

The graph displayed below
illustrates a representative test using standards with known oxalate concentrations created in CGXII
medium as their matrix. It compares both the expected values with those measured by our RevION
assay.

![](https://static.igem.wiki/teams/5829/images/oxalate-calibrationcurve.avif)

Figure 13: The calibration curve of our RevION assay matches the expected curve closely with an R2 value of 0,9692.

The mean absolute error (MAE) of this calibration set was approximately 6,82%, which is an excellent outcome for the first
successful validation. Moreover, the calibration equation achieved an R2 value of 0,9692, indicating
that the model describes oxalate concentrations with a precision of roughly 97% relative to the
measured absorbance data.

## Leaching results

---

Our leaching experiments were performed under three different conditions A, B and C with oxalate as the leaching agent.
After the leaching, the three samples and a reference samples as standard were sent to the labs of the polymer chemistry department of the university
and analyzed there via ICP.

ICP, short for Inductively Coupled Plasma, is an analytical method which relies on spectroscopy to detect several metals of interest at once,
like the PGMs rhodium, platinum and palladium in our case.

For doing so, the samples are placed into a plasma flame, which is very hot and electrically conductive. The heat disrupts all chemical bonds
and so isolating the single molecules. So the PGM oxalate complexes are destroyed and the PGMs are then free to be detected.
For detection and quantification, the PGMs are analyzed via mass spectroscopy.

Table 2: Different leaching conditions for the samples.

| Sample | A | B | C |
| --- | --- | --- | --- |
| Leaching duration [h] | 5 | 6 | 8 |

![First growth curve](https://static.igem.wiki/teams/5829/images/pgmmeasurement.webp)

Figure 13: PGM measurement of the three samples and reference via ICP-OES in mg⁄L

In the reference sample, 0,02 mg⁄L rhodium, 0,94 mg⁄L platinum and 0,19 mg⁄L palladium were detected. That means a weight percentage
of 0,03 of rhodium, 1,35 of platinum and 0,27 of palladium.

In the sample treated under condition A, 0 mg⁄L rhodium and palladium and 0,13 mg⁄L platinum were detected, which matches a weight percentage of 0,16 of platinum.

In both samples treated under conditions B and C, 0 mg⁄L rhodium and palladium and 0,03 mg⁄L platinum were detected,
which matches in both conditions a weight percentage of 0,04 of platinum.

Statistical analysis of the results revealed for all three conditions revealed a standard error of around 0,02 and a r-value of nearly one.

Regarding our results so far, the need for further optimization of our experimental set-up becomes obvious. What immediately catches the attention is that for every condition,
only one experiment and so one analysis has been performed. This of course makes our results look vague and unproven.
In the time given, we couldn't perform more experiments. In the future, we will conduct more experiments and together with our consultants in the
chemistry department improve our experimental set-up. We want to extend our set-up to specifically detect rhodium, palladium and other PGMs
and to improve our leaching and analysis of each PGM.

---

## References

1. Lange, J. et al. (2018). Harnessing novel chromosomal integration loci to utilize an organosolv-derived hemicellulose fraction for isobutanol production with engineered *Corynebacterium glutamicum*. Microb Biotechnol., 11(1), 257-263. URL: <https://doi.org/10.1111/1751-7915.12879>

2. Bell, L. (2001). The Lac repressor: a second generation of structural and functional studies. Current Opinion in Structural Biology, 11, 1, 19-25. URL: [https://doi.org/10.1016/S0959-440X(00)00180-9](https://doi.org/10.1016/S0959-440X%2800%2900180-9)

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