iGEM 2022 — Team freiburg


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# Engineering success

## Design, build, test and learn.

Much of biology is concerned with the study of phenomena and their rational explanation. Synthetic biology, however, uses these phenomena to develop new applications. Engineering and tweaking of these applications are an important part of synthetic biology. Therefore, it is sometimes better to look at the problems not as scientists but as engineers.

In the engineering cycle, you first design and model your application with the intended function in mind. Second, you build and implement the proposed construct in the designated system. Third, you perform tests and measurements regarding the effect of the construct. Fourth, you analyse and quantify your data to learn whether your application has the expected result. Finally, you return to the first step and apply your knowledge to improve the system. Repeat this DBTL cycle until you are satisfied with the performance of your application.

chAMBER is a project that tackles biosynthesis via compartmentalisation. However, it comprises a great scope of subprojects, which each underwent the DBTL cycle. We will present an iteration of the cycle dealing with the engineering and the expression of the indigo pathway and the synthetic carboxysome, the wiffleball.

![](https://static.igem.wiki/teams/4229/wiki/bmc-outlined-cropped.png)

## Design

The journey through this cycle began with the intention of proving that compartmentalising enzymatic pathways improves their yield. Therefore, we looked for different pathways to test in said compartments. After discussing several according to their complexity and the detectability of the product, we decided on a pathway to synthesise indigo, among others. Once we settled on the pathway published by Yin et al. [[1]](#egs_ref1), we codon-optimised variants of the required enzymes. We used different localisation systems to recruit the enzymes of interest into the compartments. By cloning the enzyme genes into a special pCDF –DUET-1 vector that allows the expression of several enzymes, four genes (TnaA, Fre, XiaI and TnaB) were incorporated into the two multiple cloning sites. Once in the backbone, the catcher sequences, a part of the recruitment tag, were added to TnaA and XiaI. The counterpart, simply “tags”, are added to the compartmentalisation proteins and enable the emerging enzymes to be recruited into the wiffleballs. If you want to learn more about each enzyme's exact design procedure and function, click [here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D) to get to our description page!

## Build

We built our compartmentalisation system from several components. As mentioned above, a plasmid was engineered expressing four relevant enzymes tagged with a catcher sequence. The sequences for the single enzymes were ordered from IDT and cloned into the backbone. A very important second component of chAMBER is the compartmentalisation system, which in this case was the carboxysome, or wiffleballs, which we received as plasmid from the Kerfeld group [[2]](#egs_ref2). The wiffleballs were tagged with the analogue to the catcher sequences, and both plasmids were co-transformed into competent BL21(DE3) *E. coli* to synthesise Indigo and Indirubin.

## Test

Subsequently, we tested the yield indigo from our transformed *E. coli* BL21(DE3) containing the following constructs:

* Solely the plasmid containing the indigo pathway
* the plasmid containing the indigo pathway and an empty plasmid coding only for the same antibiotic resistance as the wiffleball plasmid with the same induction (IPTG), but not the wiffleball genes themselves
* the plasmid containing the indigo pathway and the plasmid containing the genes for the wiffleball.

We prepared overnight cultures of the constructs above, inoculated them into bigger media to grow on the next day and then split them up into separate flasks to characterise and determine the best growth and induction conditions:

* Indigo plasmid induced with 0.1 mM IPTG (spectinomycin as antibiotic in the medium)
* Indigo plasmid induced with 0.04 mM IPTG (spectinomycin as an antibiotic in the medium)
* Indigo and pTrc99A plasmid induced with 0.04 mM IPTG (spectinomycin and ampicillin as antibiotics in the medium)
* Indigo and wiffleball plasmid induced with 0.04 mM IPTG (spectinomycin and ampicillin as antibiotics in the medium)
* Indigo and wiffleball plasmid induced with 0.04 mM IPTG, but only one antibiotic in the medium (spectinomycin)
* Indigo, wiffleball and ncAA synthetase together were also tested in this experiment. However, it is not relevant to this specific engineering success cycle.

The sample size for all conditions was 200 ml, 500 ml Erlenmeyer flasks were used as containers and all conditions were tested in triplicates, except for Indigo and wiffleball plasmid 0.04 mM IPTG, with only one antibiotic. This one was only tested with one sample due to the unfortunate lack of LB-medium ). All samples were supplemented with 2.5 mM L-Tryptophan and 3 mM of L-Cysteine to shift the production towards Indirubin. Samples were left to grow, and upon reaching the OD600 of 0.8, the samples were induced using the mentioned amounts of IPTG. We have decided to use 0.1 mM IPTG for the construct containing only the indigo plasmid, as previous assays have shown that this is the optimal IPTG concentration. We also used 0.04 mM IPTG for the same construct in another condition to compare it with the other constructs containing the indigo and the wiffleball or pTrc99A plasmid, as previous assays have shown that 0.04mM IPTG is the optimal concentration if both plasmids are present. We additionally tested the performance of bacteria containing both wiffleball and indigo plasmids when only one antibiotic was present to see how much a second antibiotic influences the efficiency of our pathway. Unfortunately, we could only prepare one sample for this condition. Hence, we decided to test this condition again in a later assay.

Once induced, samples were put into an incubator at 18°C while shaking at 200rpm. The assay was performed at 18°C as previous assays have shown that wiffleball formation has worked best for us at this temperature. We know that the first molecules of indirubin are produced after 24 hours, and because of that, we started to take samples of each flask after mentioned 24 hours. After that, we took new samples every six hours for the next 48 hours. Samples were collected by filling 1,5ml of each flask into separate collection tubes, which were then spun down for two minutes at 18500rpm and 4°C. The supernatant was discarded, and samples were frozen at -20°C until the assay was finished.

To evaluate our samples, pellets were resuspended in 300 µl KPI buffer and then sonicated to disrupt the cells. Afterwards, samples are spun down for 20 minutes at 18500 rpm and 4 °C. The supernatant is, again, discarded, and the pellet is resuspended with 1ml of DMSO. 200 µl of each sample was loaded on a 96-well plate and analysed in a plate reader by checking our sample's absorption at 540nm wavelength. Results were compared to a calibration curve of indirubin performed earlier to determine the indirubin concentration of each sample.

## Learn

When evaluating the essay, we found that bacteria containing fully functional wiffleball plasmids and the indigo pathway have produced higher yields of indirubin compared to bacteria that contained the plasmid with the same resistance and induction as the wiffleball plasmid. However, they still produced less than those bacteria that only contained the indigo plasmid. In conclusion, the yields produced by bacteria containing the indigo and BMC plasmids were lower than expected (see [figure 1](#egs_fig1)).

![Comparison between BL21 pCDFDuet-1 sTAF-sXTB with and without BMCs, pTrc99A plasmid-control and ncAAs.](https://static.igem.wiki/teams/4229/wiki/pictures/results/indigo/re-productionassay-3nd-bmc-comparison.jpg)

**Figure 1: Comparison between BL21 pCDFDuet-1 sTAF-sXTB with and without wiffleballs, pTrc99A plasmid-control and ncAAs.** The pathway was shifted towards Indirubin production through L-Cysteine addition. A: Schematics of the used plasmids; B: Indigo concentration measured via fluorescence (extinction 612 nm, emission 670 nm); C: Indirubin concentration measured via absorbance at 540 nm. Production with 200 mL cultures in 500 mL Erlenmeyer-flasks, at 18 °C after IPTG-induction at OD = 0.8, with L-Tryptophan concentration of 2.5 mM, L-Cysteine concentration of 3 mM, ncAA-amount of 41.24 mg pAzF and with IPTG concentration shown in legend. Photometric readout using a plate reader.

Bacteria that contained two plasmids were treated with two antibiotics, as the second plasmid codes for a different resistance. As a result, it seemed as if the bacteria with two plasmids experienced more metabolic stress and reduced the production of our target molecule. This might explain why the bacteria containing two plasmids performed worse than the ones containing only one plasmid, which in turn were only treated with one antibiotic. Our mentioned assay has confirmed this assumption, as we can see, that samples only treated with spectinomycin have produced notably higher yields than the equivalent construct/condition treated with both antibiotics. Unfortunately, we were not able to push the production above the yield of our reference construct (sTAF-sXTB 0,1mM IPTG). In conclusion, the yield improvement by the wiffleballs was still lower than expected. We inferred that this may be due to the fact that the concentrations of pathway enzymes and wiffleballs do not fit together to get the maximal number of enzymes catched while not overproducing wiffleballs. But because of both Indigo pathway and wiffleball being induced by IPTG, it was not possible for us to regulate the production of Indigo enzymes and wiffleball proteins separately from each other, limiting our experimental freedom immensely and thus, making it impossible to optimise the pathway further this way.

## Re-design

To solve this issue, we have exchanged the lac-promotor of the wiffleball plasmid with the tet-promotor which is inducible through doxycycline. This allows us to control the expression of both plasmids separately, thereby giving us the possibility to finetune the relations between wiffleball forming proteins and Indigo producing enzymes.
**image of plasmid maps with lac promotor and with tet promotor**

## Re-build

To exchange the lac-promoter for the tet-promoter we used Gibson Assembly.
![Comparison between pathway + wiffleball constructs (BMC), either with both IPTG-inducible promoter or both different promoters.](https://static.igem.wiki/teams/4229/wiki/14-promotor-change.jpg)

**Figure 2: Comparison between pathway + wiffleball constructs (BMC), either with both IPTG-inducible promoter or both different promoters.** Wiffleballs have either pLac promoter (IPTG-inducible) or tet-promoter (doxy-inducible), different doxycyclin concentrations are tested with consistent IPTG concentration of 0.04 mM. The pathway was shifted towards Indirubin production through L-Cysteine addition. A: Schematics of the used plasmids; B: Indigo-concentration measured via fluorescence (extinction 612 nm, emission 670 nm); C: Indirubin-concentration measured via absorbance at 540 nm. Production with 200 mL cultures in 500 mL Erlenmeyer-flasks, at 18 °C after IPTG-induction at OD = 0.8, with L-Tryptophan concentration of 2.5 mM and L-Cysteine concentration of 3 mM. Photometric readout using a plate reader.

## Test again

After successfully changing the promotor via Gibson assembly, we have done an assay where we checked different doxycycline concentrations while analysing their effect on the yield of our target molecules. The following conditions were tested:

* BL21 sTAF-sXTB 0,1mM IPTG
* Bl21 sTAF-sXTB + wiffleball\_tp (tet-promotor) 0,04mM IPTG, 10ng/ml Doxycycline
* Bl21 sTAF-sXTB + wiffleball\_tp 0,04mM IPTG, 20ng/ml Doxycycline
* Bl21 sTAF-sXTB + wiffleball\_tp 0,04mM IPTG, 50 ng/ml Doxycycline
* Bl21 sTAF-sXTB + wiffleball\_tp 0,04mM IPTG, 75 ng/ml Doxycycline
* Bl21 sTAF-sXTB + wiffleball (old promotor) 0,04mM IPTG

The sample size for all conditions was 200 ml, 500 ml Erlenmeyer flasks were used as containers. BL21 sTAF-sXTB 0,1mM IPTG and BL21 sTAF-sXTB + wiffleball (old promotor) 0,04mM IPTG were tested as triplicates, the remaining samples were tested only once, due to the unfortunate lack of Doxycycline. The assay and evaluation were performed the same way as stated in the previous assay above. However, we have started to take samples every six hours for 48 hours directly after induction. We have decided to do this, to save us some time, allowing us to do further experiments before wiki-freeze.

## Learn

Already during the assay, we have seen that the samples containing the new tet promotor did not produce any Indirubin. Additionally, the flasks did not appear to grow denser after induction and the pellets of our centrifuged samples showed a very light, almost white colour. Additionally, our assumption that these samples were unable to produce our target molecule was confirmed by the evaluation of this assay (see [figure 2](egs_figure2)).
Until now, we were not able to definitely determine why the change of the promotor ended in the mentioned results. Our running assumption so far is that something might have gone wrong during cloning, impeding the function of the Tetracycline resistance required for constructs that are induced through Doxycycline, resulting our bacteria kicking the bucket. However, the sequencing results for the tet promotor appeared to be correct, speaking against this assumption. We are going to look further into this, trying to understand from where this problem emerges.

## Re-design again

For the time of this writing, we were not able to make any changes to the promotor so far. However, our planning to proceed is as follows:
In case we find the reason for the inhibiting effect of the promotor, we would want to change the problematic sequence. However, in case we might not be able to find a reason, we could also exchange the entire promotor with the araBAD promotor, which is induced by Arabinose. The procedure to achieve this change would likely be by Gibson assembly.
With the then again changed wiffleball promoter, we would repeat the production assay again, testing different inducer concentrations to find out, if the yield increases and our initial thought was right, that the concentrations of pathway enzymes and wiffleball enzymes hindered better yield.

## BMCs engineering success

## Cycle 1

## Design

An important part of our project was engineering and optimizing microcompartments for bacteria. This means that the microcompartments had to be expressed in the bacteria, catch enzymes to their inside and assemble into the right conformation. The Kerfeld group, who inspired part of our work through their publication [[3]](#egs_ref3), kindly shared their wiffleball and fluorescent protein constructs with us. Initial screenings and assessments of the formed compartments seemed easiest by using fluorescence microscopy. The fluorescent proteins are both fused to a catcher (mVenus2 to the SpyCatcher, mTurquoise2 to the SnpCatcher) that specifically recognizes a tag on the internal loop of the T1 protein of the wiffleball (SpyTag and SnpTag, respectively) and binds to it, leading to the formation of a peptide bond. With this protein ligation system, the proteins can be localized inside the microcompartments.
Our initial experiments had three goals:

* show that the catching works (Fig. 3). For this we used both, untagged and tagged versions of the wiffleball T1 protein, and co-expressed them with mVenus2. Furthermore, we decided to complement our microscopy data with Western blot data (Fig. 4 + 5). For this, we took advantage of the presence of a His-tag on the T1 protein and used an anti-His antibody to detect it
* test different induction and incubation conditions to find the optimal settings to express properly formed wiffleballs

Find out the difference, if any, between minimal and full wiffleball

## Build

We prepared the samples for our initial microscopy screening by double-transforming BL21(DE3) cells with the wiffleball plasmids with or without the tags and the plasmid expressing mVenus2 also tagged.

## Test

Liquid cultures were induced with varying concentrations of doxycycline (50 and 200ng/ml) and IPTG (50 and 100µM) and cultivated for 24h at 18°C. After incubation, cells were visualized using fluorescent microscopy and harvested for western blots.

![Figure 3: Fluorescent microscopy of T1 catching the mVenus2, when the minimal or full wiffleball construct is expressed](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-3bl21-wiffleballs-comparison.jpg)

***Figure 3:**  Fluorescent microscopy of T1 catching the mVenus2, when the minimal or full wiffleball construct is expressed; A: Controls for the induction; B: T1 with and without the Spy/Snp tags; scale bar, 5µm.*

![Figure 4: Figure 1.4 in results: Western Blot showing the formation of the peptide bond between the T1 protein and mVenus2 in cells expressing the minimal wiffleball with and w/o tags](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-4-minwiffelball-tag-test-compaarision-bl21.jpg)

***Figure 4:**  Figure 1.4 in results: Western Blot showing the formation of the peptide bond between the T1 protein and mVenus2 in cells expressing the minimal wiffleball with and w/o tags. The detection of the T1 protein was performed with an anti-his antibody.*

![Figure 3: Fluorescent microscopy of T1 catching the mVenus2, when the minimal or full wiffleball construct is expressed](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-5-fullwiffelball-tag-test-comparision-bl21.jpg)

***Figure 5:**  Figure 1.5 in results: Western Blot showing the formation of the peptide bond between the T1 protein and mVenus2 in cells expressing the full wiffleball with and w/o tags. The detection of the T1 protein was performed with an anti-his antibody.*

## Learn

Through the results of this initial round of screening, we found that the mVenus2 localizes to fluorescent foci in the presence of the tagged version of the T1 protein. This localization needs the SpyTag on mVenus2. We could confirm this observation by detecting a higher molecular weight band for the his-T1 protein in the Western blot. We also observed that the minimal and full wiffleballs behave differently. In case of the full wiffleball, more fluorescent foci are found in the cells than in the case of the minimal wiffleball. Also, we observed that the combination of inducers that worked best was 100µM IPTG together with 50ng/ml doxycycline.

After evaluating our first round of experiments, we were eager to find out how to further optimize our compartmentalization system.

## Cycle 2

## Design

We opted for trying out higher IPTG concentrations to see if we could quantitatively optimize the production of the compartments.
To further enhance the system, we decided to test the wiffleballs in different strains, since the inherently different genetic backgrounds of different E. coli strains can lead to significant differences in expression and assembly of the compartments.

## Build

We transformed our plasmids into competent MG1655 cells in the same combinations as explained in cycle 1.

## Test

Liquid cultures were induced with varying quantities of IPTG (100, 400, 700, 1000µM). After 24h incubation at 18°C, cells were visualized using fluorescence microscopy (Fig. 6) and harvested for Western blots (Fig. 7). For further quantification of our data, we decided to manually count the fluorescent foci formed within the bacteria under different IPTG concentrations. While we wanted to assess the formation of the fluorescent foci(Fig. 8), we also needed to investigate the viability of the cells, since it is important for our system not to negatively impact bacterial growth. Hence, we generated growth curves for all our samples and compared them (Fig. 9). For higher reliability, we repeated these experiments three times for BL21(DE3) and twice for MG1655.

![Figure 6: Fluorescent microscopy of different induction concentrations for the BMC expression with IPTG in MG1655 mVenus2 was always induced with 50ng/ml Doxycycline](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-15mg1655-induction-comparison.png)

***Figure 6:**  Fluorescent microscopy of different induction concentrations for the BMC expression with IPTG in MG1655 mVenus2 was always induced with 50ng/ml Doxycycline; scale bar, 5µm*

![Figure 7: Western Blots of different induction concentrations for the BMC expression with IPTG in MG1655. ](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-17-mg1655-induction-experiment.png)

***Figure 7:** Western Blots of different induction concentrations for the BMC expression with IPTG in MG1655. The 100µM IPTG 0ng/ml DOX sample of the pellet and supernatant had been switched, when loading the SDS-gel.*

![Figure 8: Quantification of the foci of the full wiffleball visible under the microscope with different induction concentrations of IPTG for the BMC expression under the microscope in MG1655.](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-16-mgfoci.png)

***Figure 8:**  Quantification of the foci of the full wiffleball visible under the microscope with different induction concentrations of IPTG for the BMC expression under the microscope in MG1655; n=2.*

![Figure 9: MG1655 growth in plate reader at 30°C with different induction conditions. In legend: Wiffleball (IPTG (µM)/doxycylcine (ng/ml)).](https://static.igem.wiki/teams/4229/wiki/1-19-1-mg-growth-min.png)
![Figure 9: MG1655 growth in plate reader at 30°C with different induction conditions. In legend: Wiffleball (IPTG (µM)/doxycylcine (ng/ml)).](https://static.igem.wiki/teams/4229/wiki/1-19-2-mg-growth-full.png)

***Figure 9:**  MG1655 growth in plate reader at 30°C with different induction conditions. In legend: Wiffleball (IPTG (µM)/doxycylcine (ng/ml)).*

## Learn

The Western blot revealed that MG1655 cells barely expressed the full wiffleball. There were only few fluorescent foci in the cells. Additionally, the cells seemed to be impacted in their growth at higher IPTG concentrations. The expression of the T1 protein in the context of the minimal wiffleball construct was overall higher and detectable by Western blot. We note here that in the Western blot we can only detect (1) expression of the T1 protein and (2) whether the protein of interest (e.g. mVenus2) has been ligated to it. This assay is not the appropriate one to assess the formation of the compartments. Higher IPTG concentrations didn’t seem to influence bacterial growth. The minimal wiffleball always needed higher inducer concentration compared with the full wiffleball.
However, the change of strain did not show any significant advantage. We therefore decided to try out other strains.

## Cycle 3

## Design

The strain ME5119 is a genome-reduced derivative of MG1655 [[4]](#egs_ref4)[[5]](#egs_ref5) We hoped that, by being genome-reduced, less energy would be needed for the expression of housekeeping proteins, and therefore this strain would be more effective at synthesizing and assembling the wiffleballs. While looking for genome-reduced E. coli strains, we found another genome-reduced strain derivative of MG1655, MDS69, in particular the LowMut7 commercial version from Scarab Genomics. We decided to repeat the cycle above with the new strains.

## Build

We made competent cells of the ME5119 strain, bought competent MDS69 LowMutT7 cells from Scarab Genomics and transformed them both with the wiffleball constructs and the mVenus2 plasmid.

## Test

![Figure 10: ME5119 growth in plate reader at 30°C with different inducer conditions. In legend: Wiffleball (IPTG (µM)/Doxycylcine (ng/ml))](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-27-1-me-full.png)
![Figure 10: ME5119 growth in plate reader at 30°C with different inducer conditions. In legend: Wiffleball (IPTG (µM)/Doxycylcine (ng/ml))](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-27-2-me-min.png)

***Figure 10:**  ME5119 growth in plate reader at 30°C with different inducer conditions. In legend: Wiffleball (IPTG (µM)/Doxycylcine (ng/ml))*

![Figure 11: LowMut T7 growth in plate reader at 30°C with different inducer conditions. In legend: Wiffleball (IPTG (µM)/Doxycylcine (ng/ml))](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-29-1-lowmut-full.png)
![Figure 11: LowMut T7 growth in plate reader at 30°C with different inducer conditions. In legend: Wiffleball (IPTG (µM)/Doxycylcine (ng/ml))](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-29-2-lowmut-min.png)

***Figure 11:**  LowMut T7 growth in plate reader at 30°C with different inducer conditions. In legend: Wiffleball (IPTG (µM)/Doxycylcine (ng/ml))*

We prepared overnight cultures of the different strains and induced them with IPTG and doxycycline. Again, fluorescence microscopy was conducted, followed by a confirmatory Western blot. We also performed a growth curve assay with both strains (Fig. 10 + 11) . While we repeated all these experiments twice with ME5119, we could perform the microscopy only with MDS69 LowMutT7, due to time limitations (Western blot results will come after the wiki-freeze).

## Learn

Unfortunately, none of the genome-reduced strains showed a better result in the microscopy experiments than the BL21 strain. The fluorescent foci detected in the previous cycles could not be observed and no bands could be detected in the Western blots. However, the growth curve of the MDS69 LowMutT7 strain looked much more promising (Fig. 10 + 11), and we hypothesized that inducing and letting the cells grow at low temperatures would hinder the expression of the wiffleballs.
We therefore decided to perform another round of experiments with MDS69 LowMutT7.
By talking to Cheryl Kerfeld, an expert on BMCs and on the wiffleball in particular, whose work greatly inspired our project, we received valuable advice on changing our induction temperatures. We changed the temperature from the constant 18°C that we previously used to an initial 3 hours at 37 °C with subsequent lowering to 18 °C for another 16 hours.
Since changing the temperatures of induction and growth seemed to have great impact on the expression in experiments performed by the Kerfeld lab, we decided to also include BL21(DE3) again in our next round of experiments, given that they had initially given better results than all other strains.

## Cycle 4

## Design

We decided to investigate how this change of temperature would affect the formation of the wiffleballs, and therefore induced MDS69 LowMutT7 and BL21(DE3) with the same conditions as always. We also wanted to find out what happens when we incubate the bacteria for even longer, 48 hours in total, whether it would affect the wiffleballs (degradation, aggregation, changes in expression levels).

## Build

We used our bacteria from cycle 1 and 3.

## Test

We prepared overnight cultures of the different samples and induced them with IPTG and doxycycline. As described above, this time we started with a 3-hour incubation at 37°C and continued with 16 hours of incubation at 18°C. After 24 hours, fluorescence microscopy was conducted –and will be followed by a confirming Western blot after the wiki freeze. Afterwards, we returned the samples to the incubator and repeated the experiments after 48 hours.

## Learn

We found these conditions to dramatically improve wiffleball expression after 24 hours, registering a significant increase in the number of fluorescent foci in our bacteria in both strains. In our samples that were incubated for 48 hours, we observed even more fluorescent foci than in our previous experiments, especially in the bacteria containing the minimal wiffleball construct. In this case, the localization was not limited to the poles of the bacteria, but was more distributed throughout the cells.
We will confirm our results by Western blot before the grand jamboree.

### References

---

##### [1] Yin H. et.al., "Efficient Bioproduction of Indigo and Indirubin by Optimizing a Novel Terpenoid Cyclase XiaI in *Escherichia coli*", *ACS OMEGA*, vol.6, 20569−20576, 2021, https://doi.org/10.1021/acsomega.1c02679.

##### [2] C. Kerfeld, H. Kirst, “Bacterial microcompartments: catalysis-enhancing metabolic modules for next generation metabolic and biomedical engineering”, *BMC Biology*, vol.17, article number 79, October 2019.

##### [3] Kirst et al, “Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate”, PNAS , Vol. 119 No. 8, January 2022

##### [4] M. Hashimoto et al., “Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome,” Mol. Microbiol., vol. 55, no. 1, pp. 137–149, 2005, doi: 10.1111/j.1365-2958.2004.04386.x.

##### [5] H. Yu et al., “Minicells from Highly Genome Reduced Escherichia coli: Cytoplasmic and Surface Expression of Recombinant Proteins and Incorporation in the Minicells,” ACS Synth. Biol., vol. 10, no. 10, pp. 2465–2477, 2021, doi: 10.1021/acssynbio.1c00375.

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# Results

### Overview over the used plasmids

---

| Name | Codes for | Plasmid name | Antibiotic resistance | Promoter | Ori |
| --- | --- | --- | --- | --- | --- |
| BMC - full whiffleball with tags | H   T1 with spy- and snoopTag   T2   T3 | pHT1spysnpT2T3 | Ampicillin | IPTG-inducable PLac 0-1 | pUC |
| BMC - minimal whiffleball with tags | H   T1 with spy- and snoopTag | pHT1spysnp | Ampicillin | IPTG-inducable PLac 0-1 | pUC |
| BMC - full whiffleball without tags | H   T1  T2   T3 | pHT1T2T3 | Ampicillin | IPTG-inducable PLac 0-1 | pUC |
| BMC - minimal whiffleball without tags | H   T1 | pHT1 | Ampicillin | IPTG-inducable PLac 0-1 | pUC |
| mVenus2 | mVenus2 | pBbA2CmVenus2 | Chloramphenicol | tetR/A promotor | p15A |
| mTurquoise2 | mTurquoise2 | pBbA2CmTurquoise2 | Chloramphenicol | tetR/A promotor | p15A |
| Encapsulins | Encapsulin out of *m.Xhantus* | pMIC\_Enca | Ampicilin | IPTG-inducable PLac 0-1 | pUC |
| SPD-5 without tags | SPD-5 with spy- and snoopTag | PMIC\_spy\_SPD5\_snoop | Ampicilin | IPTG-inducable PLac 0-1 | pUC |
| SPD-5 with tags | SPD-5 | pMIC\_SPD5 | Ampicilin | IPTG-inducable PLac 0-1 | pUC |
| pAzf synthetase | pAzF aaRS/tRNA | pEVOL-pAzF | Chloramphenicol | araBAD | p15A ori |
| CNF synthetase | polyspecific MJ (M. jannaschii) tyrosyl synthetase | pULTRA-CNF | Spectinomycin | lacI | CloDF13 ori |
| pBpf synthetase | 4-Benzoyl-l-phenylalanine | pEVOL-pBpF | Chloramphenicol | araBAD | p15A ori |
| sfGFP | sfGFP | pTrc99a sfGFP   pTrc99a sfGFP G4   pTrc99a sfGFP F8   pTrc99a sfGFP W57   pTrc99a sfGFP Y74   pTrc99a sfGFP Y151   pTrc99a sfGFP D216 | Ampicilin | lacI | pBR322 |
| Liquid Droplets | SPD5::sfGFP | pBAD\_SPD5 | CmR | araBAD | p15A |
| Liquid Droplets | sfGFP | pBAD\_sfGFP | CmR | araBAD | p15A |
| Encapsulins | EncA-hisx6 | pBAD\_EncA | CmR | araBAD | p15A |
| Encapsulins | sfGFP | pET\_sfGFP | Ampicillin | IPTG-inducable T7 | pBR322 ori |
| Encapsulins | sfGFP\_TP | pET\_sfGFP\_TP | Ampicillin | IPTG-inducable T7 | pBR322 ori |
| Indigo/Indirubin pathway | XiaI with n-term SnoopCatcher   TnaA with n-term SpyCatcher   Fre   TnaB | pCDFDuet-1 sTAF-sXTB | Spectinomycin | IPTG-inducable T7 | p15A |
| Trehalose pathway | otsA   otsB | pET-Duet otsAB | Ampicilin | IPTG-inducable T7 | pUC |

* [**Rigid shells**](#re_rigid)
  + [> Wiffleball](#re_bmc_full)
  + [> Encapsulins](#re_encapsulin)

+ [**Liquid droplets**](#re_liquid)

+ [**ncAAs in *E. coli***](#re_ncaa)
  - [> ncAAs in GFP](#re_ncaagfp)
  - [> Crosslink with ncAAs](#re_ncaacross)
  - [> ncAAs in wiffleballs](#re_ncaabmc)

+ [**Pathways**](#re_pathways)
  - [> Indigo/Indirubin in *E. coli*](#re_indigo)
  - [> Trehalose in *E. coli*](#re_trehalose)

* [**Genome-reduced strains**](#re_genome)

* [**Combining systems**](#re_fullsystem)

* [**Upscaling: fedBatch & bioreactor**](#re_upscale)
* [**References**](#re_references)

## Compartmentalisation

---

## Wiffleball

**Key findings:**

* The Snp and Spy-Catchers can be used to incorporate proteins of interest into the wiffleball BMCs in different *E.coli* strains
* The assembled full wiffleball is mostly located toward the poles of bacteria, whereas the minimal wiffleball is distributed all over the cell
* The minimal wiffleball has a low metabolic burden in comparison to the full wiffleball

The synthetic BMC compartment originates from the HO-shell, which is built from five different proteins: H, T1, T2, T3, P. The synthetic full wiffleball is lacking the P-protein, which opens the shell and enables the molecules to diffuse in and out. The minimal wiffleball is the simplest form of shell, consisting only of H and T1-proteins (Figure 1.1).

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-1-bmc-schematic.jpg)

***Figure 1.1:*** Protein structure and schematic architecture of the synthetic BMC architectures compared to the native HO-shell, PDB structure 6MZX; schematics drawn with powerpoint; adapted from Kirst et al. 2022 [[1.1]](#re_ref1_1)

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/bcm.jpg)

***Table 1.1:*** Acronyms of HO-shell plasmid with their genes and their shell architecture

### **Targeting Proteins into the BMCs**

**Aim:** Testing whether the full and minimal wiffleballs form in *E. coli* and proteins are recruited inside of them.

**Experimental design:** We used fluorescent microscopy to monitor the localisation of fluorescent proteins (FPs) fused to the Spy and Snoop (Snp)-Catchers when co-expressed with the proteins required to form the full and the minimal wiffleballs, with the T1 protein being fused to the Spy and SnpTags. Specifically, the SpyCatcher is fused to mVenus2 and the SnpCatcher to mTurquoise2 (Figure 1.2). We expected that the uptake of the FPs into the compartments should alter the fluorescence distribution in the cells: instead of cytoplasmic fluorescent signal, we would expect the formation of fluorescent foci.

We used the same induced BL21 cells for the microscopy and the western blot. After an induction test, we decided to use 100µM IPTG for wiffleball induction and 50ng/ml doxycycline for the fluorescent protein expression (mVenus2 or mTurquoise2). The bacteria were grown in overnight cultures shaken at 30°C, 200rpm, induced at OD600= 0.6-0.7. Samples were harvested after 24h of incubation at 200 rpm at 18°C. Conditions were based on literature research and the initial paper from Kirst. et al. 2022 [[1.1]](#re_ref1_1). In general, culture, induction and expression conditions are highly sensitive for microcompartments since they tend to form insoluble aggregates. We also fractioned the cell lysate into supernatant and pellet to observe the solubility of the wiffleballs.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-2-spysnp-experiment.jpg)

***Figure 1.2:** Principle of catching proteins via Spy/SnpCatcher by T1 and their incorporation into the BMCs, illustrated as an example of incorporating mVenus2 in the minimal wiffleball.*

**Results:**

*All our raw data can be found on the labbook page. In this section, we only show the most salient results.*

We expressed tagged mVenus2 either alone or co-expressed with the wiffleballs, which were either tagged or not tagged with the Spy/SnpTag at the T1 protein. The results gave us important insights: samples with untagged wiffleballs show a homogeneous distribution of the fluorescence within the cells (Figure 1.3, B). When the T1 protein had the tags, we observed the appearance of fluorescent foci in some cells (Figure 1.3 B, red arrows). The foci in the full wiffleball were always found at one or both poles of the cells. The minimal wiffleball had fewer and smaller foci, which were also localised in other areas of the bacteria. The foci were easier detectable in less fluorescent cells; therefore, more foci could be hidden in cells with brighter fluorescence. BL21(DE3) showed in some induced cells an elongated phenotype (Figure 1.3 B).

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-3bl21-wiffleballs-comparison.jpg)

***Figure 1.3:*** Fluorescent microscopy of T1 catching the mVenus2, when the minimal or full wiffleball construct is expressed; A: Controls for the induction; B: T1 with and without the Spy/Snp tags; scale bar, 5µm.

We could detect the T1 protein (29 kDa w/o tags or 37 kDa with tags) on the Western Blot (Figure 1.4 and 1.5). The absence of the Spy/Snp-tag resulted in one single band. When the tag was present, a second band corresponding to circa 80 kDa was observable (Figure 1.4). The molecular weight of mVenus2-SpyCatcher is the same as that of the T protein with the tags (37 kDa). We observed the formation of the peptide bond both, with the full and the minimal wiffleball (Figure 1.4).

Always a small fraction of the unbound T1 was found in the insoluble fraction of the cells, but not when fused to mVenus2, which excludes the possibility that the nature of foci results from insoluble T1-mVenus2-aggregates. Most insoluble T1 was found in the pellet of cells expressing the minimal wiffleball, when mVenus2 was also expressed.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-4-minwiffelball-tag-test-compaarision-bl21.jpg)

***Figure 1.4:*** Western Blot showing the formation of the peptide bond between the T1 protein and mVenus2 in cells expressing the minimal wiffleball with and w/o tags. The detection of the T1 protein was performed with an anti-his antibody.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-5-fullwiffelball-tag-test-comparision-bl21.jpg)

***Figure 1.5:*** Western Blot showing the formation of the peptide bond between the T1 protein and mVenus2 in cells expressing the full wiffleball with and w/o tags. The detection of the T1 protein was performed with an anti-his antibody.

When testing SnpTag/Catcher system, we found the same kind of foci, visible under the fluorescence microscope (Figure 1.6). Therefore, we assume that we can already see the encapsulation in the BMCs and the formation of the compartment. To confirm these results, further experimental evaluation will be needed. The covalent catching of mTurquoise2 by the T1 is proven by western blots (Figure 1.7). Surprisingly, the full wiffleball showed insoluble T1 and mTurquoise2-bound T1, although only for some induction conditions. The expression of the minimal and full wiffleball does not seem to influence the formation of insoluble aggerates of the proteins and seems to happen in a more random fashion.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-6bl21-venusundturquoise-comparison.png)

***Figure 1.6:*** Representative fluorescence microscopy images of BL21 cells transformed with the indicated constructs. A: Controls; B: T1 with and without the Spy/Snoop tags. Scale bar, 5 µm.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-7bl21-mturquoise.png)

***Figure 1.7:*** Western Blot showing the formation of the peptide bond between the T1 protein and mTurquoise2 in cells expressing the full and the minimal wiffleball with and w/o tags. The detection of the T1 protein was performed with an anti-his antibody.

**Conclusion:**

Taken together, we believe that the FPs are being encapsulated into the wiffleballs, which form in the *E. coli* cells we used. A more direct proof of the formation of the compartments and the localisation of the FPs into them is, however, needed.

### **Strain comparison for testing the orthogonality of wiffleball expression**

**Aim:** Testing different *E. coli* strains for their ability to support the formation of the compartments monitoring their respective burden.

**Experimental design:** We tested the full and minimal wiffleball, besides in BL21(DE3), in MG1655, the standard K-12 *E. coli* strain and in C321∆exp, a strain engineered for more efficient incorporation of non-canonical amino acids into proteins of interest. In this strain all the instances of the amber STOP codons have been replaced with other STOP codons and Release Factor 1 has been deleted. The experimental conditions stayed the same as in the previous experiment for the Spy/SnpTag/Catcher testing.

**Results:**

We first tested the BMCs with the SpyTag/Catcher system catching mVenus2 in MG1655. Under the fluorescent microscope we were able to observe the same foci like in BL21(DE3), but in lower quantities (Figure 1.8).

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-8mg1655-wiffleballs-comparison.png)

***Figure 1.8:** SpyTag/Catcher system in MG1655, when the minimal or full BMC construct is expressed; A: Controls for the induction; B: T1 with and without the Spy/Snp tags; scale bar, 5 µm.*

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-9-wb-mg1655-min-spycatch.png)

***Figure 1.9:** Western Blot of the BMC minimal wiffleball (pT1spysnp) + mVenus2 in MG1655.*

Next, we repeated the same experiments under the same conditions in C321∆exp. We did not see any signal on the Western Blot corresponding to the T1 protein when using the anti-His antibody in cells expressing both, the full and the minimal wiffleball. The loading control was still observable (data found in the supplementary). Cells expressing the full wiffleball showed one fluorescent focus per 100 cells when observed under the microscope (data found in the supplementary).

**Conclusion:**

The expression of the wiffleball proteins in MG1655 and in C321∆exp is not easily achievable. A problem could be the protein synthesis at 18°C. On the other hand, the use of higher temperature is known to result in unproper compartment formation. The expression and correct formation of BMCs is an often-claimed challenge in publications.

### **Induction experiments for improvement of wiffleball expression**

**Aim:** Enhancing the protein expression level to an optimal balance between high enough expression and low metabolic burden.

**Experimental design:** We tested different inducer concentration and validated the expression with fluorescence microscopy for the foci formation, western blot for the protein expression level and growth assays for the metabolic burden. The experimental conditions for the microscopy and the western blotting stayed the same. For the growth assay, the bacteria were induced at OD600= 0.1 in a 96-well plate with all the different inducer concentrations and incubated at 30°C, shaken in a plate reader for 20h. These conditions were changed due to the lack of a cooling function to 18°C of the device, limited time of the availability of the plate reader and a restricted maximum growth to OD600= 1.4 due to the 200µl well volume. For comparison between the strains, both strains were measured on the same 96-well plate.

We tested the full and minimal wiffleball in BL21(DE3) and MG1655 with different concentration of IPTG reaching from 100µM (best induction condition from the first induction test) up to 1mM.

**Results:**

**BL21(DE3)**

In the microscopy, the number of foci increased with the amount of the inducer. Expressing the minimal wiffleball with a concentration of 400µM IPTG and higher, resulted in many small foci across the whole cell. This can be easier observed in less fluorescent cells, where the foci can be better distinguished from the fluorescent background (Figure 1.10). An exact quantification for this is not possible due to the bright, overlaying fluorescent signal. Again, the longer phenotype occurred in the minimal wiffleball. For the full wiffleball we quantified the bigger foci at the poles per number of bacteria. Compared to the minimal wiffleball, they can be still observed in presence of high fluorescent background (Figure 1.11). The mean percentage of foci per cell number was merely constant around 52%. An induction with 1mM effected in a drop of 8%.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-10-bl21-induction-comparison.png)

***Figure 1.10:** Fluorescent microscopy of different induction concentrations for the BMC expression with IPTG in BL21(DE3), mVenus2 was always induced with 50ng/ml Doxycycline; scale bar 5µm.*

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-11-quant-foci-bl21-full.png)

***Figure 1.11:** Quantification of the foci of the full wiffleball visible under the microscope with different induction concentrations of IPTG for the BMC expression in BL21(DE3); n=2*

The western blots showed similar results like the microscopy data (Figure 1.12). For the induction with 100µM and 400µM IPTG we could detect an insoluble part of the T1 and T1-mVenus2 in the pellet of the full wiffleball. We quantified the signal for the bands at the expected molecular weight for T1 and T1-mVenus2 and normalised it to the signal of the loading control (RNA polymerase b subunit) and have been quantified by the area under peak method. The quantification showed that the best inducer concentration was 400µM for the full wiffleball and 700µM for the minimal wiffleball (Figure 1.13). The Western Blots were repeated three times, but not all membranes were able to be quantified due to overlapping bands or curved SDS-PAGE runs.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-12-wb-bl21.png)

***Figure 1.12:*** Western Blot used to quantify the expression levels of the T1 protein and the amount of T1-mVenus2 ligation product for the indicated IPTG concentrations in BL21(DE3) cells expressing the minimal and the full wiffleball. The detection of the T1 protein was performed with an anti-his antibody.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-13-1westernblot-quantification-bl21-full.png)
![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-13-2westernblot-quantification-bl21-min.png)

***Figure 1.13:*** Quantification of the Western Blots shown in Figure 1.12.

For the growth assay in BL21(DE3) we could find different, but not constant trends. First, BL21(DE3) grew the best with empty plasmids, followed up by the minimal and the full wiffleball, which seemed to affect the growth the most (Figure 1.14). We repeated the growth assay experiment three times, but the results seemed to be influenced randomly. We could not determine a major trend in growth by the expression of our synthetic constructs. Overall, BL21(DE3) seems not be highly burdened by expressing the wiffleball.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-14-bl21-growth.png)

***Figure 1.14:** Growth comparison between BL21(DE3) and MG1655 with induction of 100µM IPTG 25ng/ml Doxycycline, n=1*

In MG1655, there were remarkably fewer foci in cells expressing both, the full and the minimal wiffleball (Figure 1.14). Quantification of the number of foci in cells expressing the full wiffleball showed a range from 18% to 30% of foci per number of cells, with an increase at 400µM IPTG to 26% (Figure 1.15).  We did not quantify the cells expressing the minimal wiffleball because of the too high background fluorescence.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-15mg1655-induction-comparison.png)

***Figure 1.15:** Fluorescent microscopy of different induction concentrations for the BMC expression with IPTG in MG1655 mVenus2 was always induced with 50ng/ml Doxycycline; scale bar, 5µm*

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-16-mgfoci.png)

***Figure 1.16:** Quantification of the foci of the full wiffleball visible under the microscope with different induction concentrations of IPTG for the BMC expression under the microscope in MG1655; n=2.*

The western blot of the same microscopy samples showed, as in the prior data, no bands of the T1 proteins.  After increasing the sensitivity, slight band of bound and unbound T1 could be detected in the full wiffleball construct (Figure 1.17). The protein is still expressed, which correlates with the microscopy data. It could be assumed, the detection range of the western blot of the first test of the SpyTag/Catcher in MG1655 was just too low. The foci seem to already be formed at low BMC protein concentrations. An insoluble fraction of the protein wasn’t found. The western blots were repeated 3 times.

The minimal wiffleball showed distinct bands compared to the full wiffleball. The protein expression seems to be overall higher compared to the full construct. Less diverse proteins results in less metabolic burden. Surprisingly, in Figure 18 the 700µM induction had the most bound T1-mVenus2 fraction. In the repetition of the Western Blot, the bound T1 appeared stronger than the unbound compound. These conditions should be treated carefully, because overexpressing the wiffleball can result in not proper forming of the compartment.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-17-mg1655-induction-experiment.png)

***Figure 1.17:** Western Blots of different induction concentrations for the BMC expression with IPTG in MG1655. The 100µM IPTG 0ng/ml DOX sample of the pellet and supernatant had been switched, when loading the SDS-gel.*

![](https://static.igem.wiki/teams/4229/wiki/1-18-wb-quant-mg1655.png)

***Figure 1.18:** Quantification of the Western Blots shown in Figure 1.16 only for cells expressing the minimal wiffleball.*

The growth assay highlighted that the induction conditions of 100µM IPTG + 25ng/ml doxycycline did not affect the growth of MG1655 (Figure 1.19). As we expected, the full wiffleball had more negative impact on the growth than the minimal wiffleball, which consists of less diverse proteins.

![](https://static.igem.wiki/teams/4229/wiki/1-19-1-mg-growth-min.png)

![](https://static.igem.wiki/teams/4229/wiki/1-19-2-mg-growth-full.png)

***Figure 1.19:** MG1655 growth in plate reader at 30°C with different induction conditions. In legend: Wiffleball (IPTG (µM)/doxycylcine (ng/ml)).*

Comparing the growth of both strains in the same plate reader, MG1655 is growing faster and does not die after 10h like BL21(DE3) (Figure 1.20). However, the protein expression of MG1655 cannot be related to the comparable high expression level of BL21(DE3), which affects the metabolic burden, therefore resulting in less growth.

For more data of the growth check our supplementary data.

![](https://static.igem.wiki/teams/4229/wiki/1-20-growth-comparison-bl-mg.png)

***Figure 1.20:** Growth comparison in plate reader at 30°C between BL21(DE3) and MG1655 with induction of 100µM IPTG 25ng/ml doxycycline.*

**Conclusion:**

Overall, the metabolic burden of the cell influenced than less then estimated. With an inducer concentration of 100µM IPTG, the cells are hardly affected in growth. The minimal wiffleball shows in general better expression and foci forming at higher inducer concentration and should be therefore induced with higher IPTG concentrations than the full wiffleball.

**Late protein expression and degradation**

**Aim:** Does MG1655 not express the compartment at all, or could it be possible that it gets degraded before we can even detect it? Does this strain just need more time to produce the proteins instead?

**Experimental design:** The already used induction conditions for the wiffleball induction at OD600= 0.6-0.7 with incubation at 18°C, 200rpm, stayed the same. We took samples from the induced cultures from different time points, after 19h, 24h and 48h from MG1655. We examined the foci formation and protein expression by fluorescent microscopy and western blotting as in the wiffleball experiments before.

Furthermore, to investigate whether harvesting the samples earlier than the prior 24h induction has a remarkable effect on our protein expression and BMC formation, the 19h and 24h time points of induced BL21(DE3) were tested by western blotting.

**Results:**

MG1655 showed a slight decrease in foci formation after 48h compared to the 24h incubation (Figure 1.21). The incubation between 19h and 24h barely had any influence, detectable by qualitative fluorescence microscopy observation.

After 24h of incubation, BL21(DE3) had a higher T1 expression than the 19h sample.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-21-mg1655-time-comparison.png)

***Figure 1.21:** Fluorescent microscopy after different incubation times for the wiffleball expression in MG1655 with 100µM IPTG for BMCs and with 50ng/ml Doxycycline for; scale bar, 5µm.*

**Conclusion:**

Longer incubation times had nearly no effect on the overall protein expression of MG1655.

**Is the wiffleball only expressed at the poles or in the whole cell?**

**Immunostaining for BMC distribution:**

**Aim:** We still don’t know if the wiffleball is formed properly. We are certain that the fluorescent spots do not represent inclusion bodies but rather compartment structures, judging by the investigation of the insoluble cell fraction. To determine their distribution in the cell and the right formation of the compartments, electron microscopy would be the best option. However, due to the high effort and limited use of this advanced technique, we first want to detect the overall distribution and formation of the wiffleball. We chose detection *via* immunofluorescence microscopy, even if this method is less established in bacteria.

**Experimental design:** For this experiment, BL21(DE3) bacteria were induced at OD600 = 0.6-0.7 with 100µM and 400µM IPTG, additionally with and without 50ng/ml doxycycline for mVenus2 expression, and subsequently incubated at 18°C for 18h, shaking at 200 rpm (the same way as in the previous experiments). After fixation with paraformaldehyde, the cells were treated with 70% ethanol to reduce the cell shrinking caused by the fixation. Next, the cells were immobilised in poly-L-Lysine coated chamber slides and treated with lysozyme and DNAse, for optimal diffusion of the antibodies through the cells. For the staining, our previously applied anti-His-antibody, which binds the T1, was used. The secondary anti-mouse antibody was linked to fluorescent stain DyeLight 650 (Invitrogen sponsoring, Goat anti-Mouse IgG (H+L) Secondary Antibody, DyeLight 650), which fluoresces in the far-red spectrum and does not interfere with the mVenus2 emission.

**Results:**

We could prove that the full wiffleball is still forming foci at the poles in absence of mVenus2 (Figure 1.22 A, Full wiffleball +IPTG). Additionally, more round structures were highlighted by the anti-His DyeLight650. A fluorescent background in the bacteria was also observable, which could be interpreted as unbound T1. In the minimal wiffleball these round structures could be observed as well, mostly distributed over the whole cytoplasm. The specificity of the anti-His antibody was shown in the negative control, when only mVenus2 was expressed. No fluorescent signal could be detected in the far-red channel. (Figure 1.22 A +DOX)

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-22-bl21-immunofluorescence.png)

***Figure 1.22:** Immunofluorescence staining with anti-His-antimouse-DyeLight650 conjugate, full wiffleball induced by 100µM IPTG, minimal wiffleball induced by 400µM lPTG, mVenus2 induced by 50ng/mFl doxycycline: A: controls and wiffleball expression in absence of mVenus2; B: anti-His staining in presence of mVenus2,; scale bar, 5µm.*

**Conclusion:**

In comparison to the previously estimated fluorescence microscopy experiments, the immunostaining showed that T1 is spread all over the cell and a lot more structures that seemed to be assembled wiffleballs could be observed. Ultra-resolution microscopy has determined the roundish structures caused by the wiffleballs, with two distinct types depending on the size. The bigger foci often polarised in the full wiffleball, and the smaller, more distributed structures, in the minimal wiffleball.

**Visualizing the wiffleball formation at highest scale:**

**Aim:** To push the structural resolution to the next level, electron microscopy was used to gain insights into the wiffleball structure and distribution in the cells in the highest resolution possible.

**Experimental design:** We induced BL21(DE3) with four conditions: empty backbone as control, full wiffleball w/o any incorporated cargo (100µM IPTG), full wiffleball with mVenus2 (100µM IPTG + 50ng/ml doxycycline) and the full wiffleball with our incorporated enzymes (150µM IPTG; the wiffleball and the indigo enzymes were induced by IPTG). After 24h of incubation at 18°C, 200rpm, we delivered the samples to Marta Rodriguez, the head of the TEM facility of the university of Freiburg. With her help, we could observe the four conditions of the full wiffleball under the TEM. The samples were prepared by negative staining with osmium tetraoxide and uranylacetate.

**Results:**

After a thoroughly inspection of all the pictures taken by the TEM, we noticed that in all the samples where the microcompartment has been induced, darker regions at the cell poles (or sometimes in the middle of the cell) are forming (Figure 1.23, light blue arrows). This observation correlates to the dots we detected under the fluorescence microscope. The dark regions are probably too big to directly be microcompartments, however we suppose they are colocalisations of microcompartments, which would mean that the wiffleballs gather at the cell poles. To be able to certainly affirm this hypothesis, we would need an immunogold labeling to mark the exact position of the constructs. Previous research already showed the localisation of bacterial microcompartments in darker spots at the cell poles, even with the immunogold labeling technique [[1.2]](#re_ref1_2), which represents a great hint for the veracity of our work.

By analyzing the poles and the darker spots more closely, we discovered some other, smaller, interesting structures (Figure 1.23 red arrows, Figure 1.24). In the bacteria containing an empty backbone we saw some circular texture that looked to us like air bubbles, maybe generated due to the preparation of the sample. In the bacteria with the expressed wiffleball, we found some circular structures that might indicate the formation of a microcompartment. We noticed something that looks like a multi-layered element, containing what seems to be particles on its inside. A similar observation was already described by other research groups in the past [[1.2]](#re_ref1_2)[[1.3]](#re_ref1_3) which makes everything even more intriguing.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-23-tem-finalpic.png)

***Figure 1.23: Transmission electron microscopy.**  Darker regions at the cell poles can be observed where the wiffleball is induced.*

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-24-tem-structures-maybebmcs.png)

***Figure 1.24: Transmission electron microscopy.**  closer look at the cell structure showing possible formations of the full wiffleball; in the empty backbone, a structure is shown which can be differentiated from the others, but was highlighted for comparison.*

**Conclusion:**

The results look promising, but for a definite proof the immunogold labeling method should be applied. We still have upcoming experiments with the electron microscopy facilities to investigate the wiffleballs even further.

**Minimal wiffleball affecting the cell morphology**

Along the course of our experiments with BL21(DE3), we noticed that in the samples where the minimal construct of the wiffleball was induced, the cells were very often elongated, sometimes even “spaghetti-like”, reaching the length of over 100µm, as you can see in Figure 1.25. This leads to the conclusion that the expression of this microcompartment probably hinders the cell division, letting the bacteria grow more than usual. It would be interesting to see, through a DNA staining/screening, whether the DNA is evenly distributed along the whole cell, at only one or two poles of the cell, or aggregated in many parts of the organism, maybe indicating the attempt of the bacterium to divide.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-25-bl21-longbacteria.png)

***Figure 1.25: BL21(DE3) alterated phenotype by expressing the minimal wiffleball.** Microcompartment constructs induced with 400µM IPTG, mVenus2 with 50ng/ml doxycycline. Scale bar: 20µm.*

**Improving the interaction between XiaI and TnA and the T1 protein:**

**Aim:** To adjust and better control the wiffleball and indigo enzyme interaction, we decided to switch the promotor of all wiffleball constructs from the IPTG-induction to the Tet-induction system from the mVenus2 promotor. In this way, the concentration of the pathway and compartment protein can be optimised, next to the temporal expression, to ensure that more enzymes are caught by T1, and less empty compartments are present.

**Experimental design:** We switched the promotor by Gibson assembly, where we amplified the wiffleball backbone of every construct by itself and used base pair extension on the Tet-operator and Tet-promotor sites for an 15bp overlap to the backbone. The success of the cloning was proved by colony PCR and a followed sequencing of the Tet-insert.

The sequencing result confirmed the correct cloning. After the induction of transformed BL21(DE3), the cells showed a reduction in growth. With the brightfield microscopy, it became clear that the cells were unhealthy or dying when the plasmid was induced and not affected by the presence of doxycycline itself, as seen in our control. The western blot showed a slight protein band in the full wiffleball and thin bands in the minimal wiffleball. No correlation between the inducer concentration and the band thickness was found.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-26-tet-switch-alternative-to-plasmid-map.png)

***Figure 1.26:** Upper plasmind: plasmid with exchanged Tet promotor and operator. Bottom plasmid: previous expression under lacI regulated operator.*

## Encapsulins

**Key findings:**

* Expression of sfGFP containing a targeting peptide in the presence of encapsulins cause formation of fluorescent "foci" observable with fluorescent microscopy
* Addition of targeting peptide on sfGFP does not hinder fluorescence or correct folding
* Further experiments are needed to confirm the formation of correctly formed encapsulins

**Aim:** our goal was to use the encapsulin protein EncA from *M. xanthus*, express it heterogeneously to form nanocompartments in *E.coli* . We want to use these encapsulins nanocompartments as chassis for enzymatic reactions to colocalise individual enzymes of a given pathway. Proteins are targeted to the Encapsulins due to a targeting peptide (TP), which is present C-terminal on the cargo protein. Therefore, we cloned two versions of the sfGFP, one without any targeting peptide and one with the targeting peptide C-terminally. In this way we can easily distinguish whether the sfGFP is incorporated into the Encapsulins with fluorescent microscopy.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/plasmids-enca-1.png)

**Figure 2.1:** The Encapsulin gene was cloned into pBAD33 and the sequence of sfGFP was cloned into a pET plasmid which are induced with arabinose and IPTG, respectively.

A plasmid containing a sfGFP with the same sequence as the biobrik # was used as a template PCR to extend the sequence with a linker and a targeting peptide (TP) on the C-terminus. This targeting peptide is responsible for the targeting of the native cargo into the Encapsulin of m. Xanthus. In this case we clone it onto sfGFP for direct detection of encapsulation with fluorescence microscopy. The sequence of the targeting peptide has been codon optimised for E. coli expression.

**Sequence of GS-linker:** GGCGGCGGCGGCAGC

**Sequence of targeting peptide:** CCGGAAAAACGTCTGACCGTGGGCAGCCTGCGTCGTTAA

The Encapsulin protein EncA, was first codon-optimised for *E.coli* from the sequence from the NCBI with the entry: MXAN\_3556. We ordered the sequence optimised version from IDT containing a linker and a his6-tag on the C-terminus.

**Sequence of the GS-linker and his6-tag:** AGTGGCAGCGGCCACCACCACCACCACCAC

**Experimental setup:** BL21 were transformed with pBAD\_encA and either pET\_sfGFP or pET\_sfGFP\_TP. A larger culture was grown until OD: 0.6-0.8 and induced with arabinose for the Encapsulins and IPTG for the sfGFP. Then cells were incubating for 24 h at 18°C until samples were analyzed with fluorescent microscopy.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-enca-02.png)

***Figure 2.2: Fluorescence microscopy of Encapsulin expression.*** *E. coli (BL21) was co-transformed with EncA and either A) sfGFP or A) sfGFP C-terminally fused to the targeting peptide for the Encapsulin. Arabinose and IPTG induces the expression of EncA and sfGFP respectively. Panels presents GFP fluorescence. sfGFP localises mostly throughout the cytoplasm in both conditions. Single dots in cells are formed when IPTG is only expressed due to leaky expression. Representive pictures of* *three replicates.*

Fluorescent microscopy of the Encapsulins with target peptide showed for all conditions with 100 µM IPTG induction of sfGFP occasionally fluorescent “foci”. When sfGFP is only present due to leaky expression, a larger population of the cells expressing the sfGFP with targeting peptide have visible fluorescent dots. The results were surprising, as we expected the encapsulins to be formed when sfGFP was induced and not before. The absence of fluorescent dots when sfGFP\_TP with targeting peptide we hypothesise is related to an overexpression of sfGFP\_TP in comparison to encapsulins. The amount of sfGFP is likely too high and not all proteins gets targeted to the encapsulins and as a result distribute through the cytoplasm. In theory, every EncA protein could bind a cargo protein, however, the actual number of cargo proteins are found to be 24% [[2.1]](#re_ref2_1).

Because some fluorescent dots were observed even in the absence of encapsulins for both conditions, we performed a control experiment with the two types sfGFP and no encapsulins.

**Aim:** The aim is to observe whether the observed fluorescent dots for the experiments with Encapsulins are due to leaky expression the Encapsulins.

**Experimental setup**: BL21 was transformed with pBAD33, same backbone as for the Encapsulins, and a plasmid containing either sfGFP or sfGFP with the encapsulation tag C-terminally.  The induction and incubation follow the same procedure as for the previous experiment.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-enca-03.jpeg)

**Figure 2.3: Fluorescence microscopy of Encapsulin expression.** *E. coli (BL21) was co-transformed with pBAD33 and either A) sfGFP or A) sfGFP C-terminally fused to the targeting peptide for the Encapsulin. Arabinose and IPTG induces the expression of pBAD33 and sfGFP respectively. Panels presents GFP fluorescence. sfGFP localises mostly throughout the cytoplasm in both conditions. Single dots are formed occasionally.*

Figure 2.3 shows that expression of both the sfGFP alone and sfGFP fused C-terminally to the encapsulation tag mostly leads to localisation throughout the cytoplasm. However, for both strains, occasional dots with higher fluorescent intensity are formed. These fluorescent dots have similarities to those observed when expressed together with Encapsulins. As these dots are more commonly seen when sfGFP is expressed in the presence of the Encapsulins, this could be due to the generally higher protein production causing formation of inclusion bodies. With this experiment, however, we can also conclude that both the normal sfGFP as well as sfGFP fused to targeting peptide can form fluorescent dots, which may be difficult to distinguish from the encapsulated sfGFP. Furthermore, the sfGFP containing the targeting peptide show similar characteristics as the normal sfGFP.

As the interaction between the targeting peptide and the encapsulin protein is non-covalent, it can not be detected as a shift on a western-blot. However, we decided to blot the encapsulins with an antibody against the his6-tag.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-enca-05.png)

**Figure 2.4: Western blot of Encapsulin expression.** *E. coli (BL21) was co-transformed with EncA and  sfGFP C-terminally fused to the targeting peptide for the Encapsulin. Arabinose and IPTG induces the expression of pBAD33 and sfGFP respectively. Panels presents GFP fluorescence. EncA expression is at very low levels and is detected in supernatant as well as pellet. Encapsulins are detected using anti-his antibody, loading control is the RNA polymerase ß-subunit.*

Figure 2.4 shows a western blot for the expression of EncA. The signal from EncA was almost not detectable and so the brightness has been changed to show the presence of the bands. The marker is added seperately from the same membrane. There is a faint band for the EncA at about 35 kDa which is close to the expected size (37 kDa). The western blot supports the results seen in the microscope, that EncA is relatively low expressed and this could be the reason why we only see encapsulation at low concentrations of sfGFP.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/plasmids-enca-1.png)

**Figure 2.5: Plasmids of encapsulin and sfGFP in changed backbone.**

We cloned our EncA into the same as the microcompartment and the SPD-5 (pBbE6a). We also changed the backbone of the sfGFP containing the C-terminally fused target peptide to the same as the mVEN (pBbA2c). We repeated the microscopy results seen for the encapsulins in the pBAD33 backbone.

**Experimental setup**: BL21 was transformed with pBAD33, same backbone as for the Encapsulins, and a plasmid containing either sfGFP or sfGFP with the encapsulation tag C-terminally.  The induction and incubation follow the same procedure as for the previous experiment.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-enca-041.jpeg)

**Figure 2.6: Fluorescence microscopy of Encapsulin expression.** *E. coli (BL21) was co-transformed with sfGFP C-terminally fused to the targeting peptide for the Encapsulin. and either A)  or B) EncA  IPTG and Doxycyclin induces the expression of the EncA/ and sfGFP\_TP respectively. Panels presents GFP fluorescence. sfGFP localises to fluorescent dots when expressed with encapsulins (scale bar 5 µm).*

Figure 2.6 shows that the expression of sfGFP was generally very low and mostly not detectable. However, when expressed in the presence of the encapsulins the formation of fluorescent dots could be observed. These results are similar to the previous where even leaky expression of sfGFP led to the formation of fluorescent dots.

From these results, it is clear that the formation of fluorecent “foci” is related to the expression of the encapsulins, but also that similar structures occasionally can occur even in the absence of encapsulins. The encapsulation *via* the targeting peptide is, as mentioned, non-covalent and as a result would be possible to be detected on native PAGE.

**Aim:** The encapsulins in their native state are expected to run as >5 MDa and wouldn’t be able to enter far into a native PAGE gel due to its size [[2.1]](#re_ref2_1). Successful encapsulation of sfGFP containing the targeting peptide would be detected in these bands.

**Experimental setup**: BL21 were transformed with pBAD\_encA and either pET\_sfGFP or pET\_sfGFP\_TP. A larger culture was grown until OD: 0.6-0.8 and induced with arabinose for the Encapsulins and IPTG for the sfGFP. Then cells were incubating for 24 h at 18°C until samples were analyzed with fluorescent microscopy. Prior to microscopy, 3 ml liquid culture is pelleted and lysed for the native PAGE.

Figure 2.8 shows the native PAGE gel fluorescent signal. Figure 2.7 shows the microscopy pictures of the same samples for comparison. It is important to note that while 3 ml of each sample was used for the preparation for the native page it is not possible to fully compare the fluorescent signal betwween the samples, as there is no loading control. For all samples there is some signal detected, corresponding to the expression of sGFP. This is also the case, even in the absence of induction. However, leaky expression of sfGFP is also detected in fluorescent microscopy. In the supernatant, fluorescent signal could be detected for conditions where sfGFP or sfGFP\_TP was induced, even in the absence of encapsulin induction. Based on the microscopy results, the fluorescent “foci” were only detected for the sfGFP\_TP strain and so this is an unexpected result. This would suggest that the presence of the targeting peptide does not influence the occurence of these structures. Furthermore, if these bands represent the encapsulins we would only expect them to be formed when encapsulins are induced. Therefore, it is difficult to conclude on the occurrence of fluorescent “foci” in the microscopy pictures and the native page results. For further proof of the correct formation of encapsulins methods such as Electron Microscopy (EM) would be nessesary. However, due to time constraint, this was not possible.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-enca-10.png)

**Figure 2.7: Fluorescence microscopy of Encapsulin expression.** *E. coli (BL21) was co-transformed with EncA and either A) sfGFP or A) sfGFP C-terminally fused to the targeting peptide for the Encapsulin. Arabinose and IPTG induces the expression of EncA and sfGFP respectively. Panels presents GFP fluorescence. sfGFP localises mostly throughout the cytoplasm in both conditions. Represen**tive pictures of Native PAGE samples.*

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-enca-09.png)

**Figure 2.8: Native PAGE of encapsulins.** *E. coli (BL21) was co-transformed with either sfGFP or sfGFP C-terminally fused to the targeting peptide for the Encapsulin, and EncA. The plasmids were induced as described in the table. Fluorescence signal of GFP channel is shown in dark. Signal from sfGFP is present for all samples, however only in the supernatant is a fluorescent signal in the bottom of the wells detected.*

### Conclusion

Through this project, it was possible to express and detect the formation of the encapsulin-forming protein, EncA. Furthermore, results of fluorescent microscopy suggest the formation of fluorescent “foci” is related to the expression of EncA together with the targeting peptide containing sfGFP. However, occasionally a few similar structures could be found for other conditions. Native PAGE results suggest the formation of a larger structure in the supernatant, however, in contrast to the microscopy, this is also present without EncA expression and also for sfGFP with no targeting peptide. As mentioned, further repetitions and experiments would be needed to confirm these findings.

## **Liquid droplets: SPD-5**

**Key findings:**

* Expression of SPD-5 fusion protein with sfGFP cause formation of fluorescent "foci" observable with fluorescent microscopy
* SPD-5::sfGFP "foci" can show dynamical characteristics, expected for liquid droplets
* SPD-5 fused N- and C-terminally to spy- and snoop-tag are perform cathing of catcher containing fluorescent proteins.

The sequence of the SPD-5 from C. Elegans was codon-optimised for expression in E. coli and ordered in two parts from IDT. Gibson assembly was performed for the cloning into a pBAD33 backbone already containing the sequence for sfGFP. The fusion of SPD-5 to sfGFP allows for the visualisation with fluorescent microscopy.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/plasmids-spd5-1.png)

**Figure 3.1:**  Plasmids schemes of SPD-5 construct and sfGFP in pBAD

**Aim:** the aim of the first experiment was to test the SPD5::sfGFP construct with microscopy, with our newly cloned construct.  As it is a fusion construct the fusion of SPD-5 to another protein at the C-terminus could cause the proteins to misfold.

**Experimental setup**: Strains are prepared in BL21 and are transformed with either pBAD33 containing sfGFP or SPD5::sfGFP and induced with either 0, 0.005 or 0.1% arabinose at OD: 0.6-0.8, then incubated for 24h at 18°C.

Figure 3.2 shows that the fluorescent signal is dispersed throughout the cytoplasm when sfGFP is expressed alone and the occurrence of "foci" with strong intensity when it is expressed as a fusion protein with SPD-5. However, the negative control of the fusion protein appears to equally express the protein of interest and form similar dots. The plasmids were later sequenced for mutations in the promoter region, however, no differences were found between the plasmid containing sfGFP alone and the SPD-5::sfGFP fusion protein.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-spd5-01.png)

**Figure 3.2: Fluorescent microscopy of SPD-5::sfGFP expression.** BL21 was transformed with either. A: pBAD33 containing sfGFP or B: SPD-5::sfGFP fusion protein. Increasing concentrations of inducer results in stronger fluorescence. Bacteria expressing SPD5::sfGFP form dots with strong fluorescent intensity. The images present representable regions of two experiments (scale bar 5 µm).

Phase-separation droplets are expected to be dynamic, however, the dots observed did not move over time or fuse with other droplets. The experiment was repeated twice with similar results. Previous experiments have described how liquid droplet forming proteins transition from a dynamic state, where the droplets can move inside the cell to a more static gel-like phase [[3.1]](#re_ref3_1). There is a clear difference in the structures formed between the induction concentrations with higher concentrations leading to undefined large areas of fluorescence. Similar results have been shown for constructs where different arrangements of hydrophilic regions were tested [[3.1]](#re_ref3_1). The observations could be due to protein overexpression leading to inclusion bodies formed at the poles or that SPD-5 droplets are in their gel-like state. As BL21 is a strain optimised to produce large amounts of heterologous protein we decide to test our constructs in MG1655, a reference K-12 strain.

**MG1655**

**Aim:** the aim of the first experiment was to replicate the SPD5::sfGFP microscopy results, in MG1655 a standard K-12 *E.Coli* strain.

**Experimental setup**: Strains are prepared with MG1655 and are transformed with either pBAD33 containing sfGFP or SPD5::sfGFP and induced with either 0, 0.005 or 0.1% arabinose at OD: 0.6-0.8. Then incubated for 24h at 18°C.

Figure 3.3 shows the expression of the cloned constructs in MG1655 with fluorescent signal dispersed over the cytoplasm when sfGFP is expressed alone and the occurrence of areas with strong intensity when it is expressed as a fusion protein with SPD-5. Again, the uninduced sample of SPD5::sfGFP shows a fluorescent signal. For this experiment a lower concentration of Arabinose was tested, 0.001%, which also showed the ability to form fluorescent "foci".

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-spd5-02.png)

**Figure 3.3: Fluorescent microscopy of SPD-5::sfGFP expression.** MG1655 was transformed with either A: pBAD33 containing sfGFP or B: SPD-5::sfGFP fusion protein. Panels show GFP fluorescence. Bacteria expressing SPD5::sfGFP form dots with strong fluorescent intensity. The images present representable regions of two experiments (scale bar 5 µm).

In contrast to the previous results, this time there was obvious movement of the fluorescent dots. Meaning, they changed position inside the cells independently of cell movement. This type of movement was recorded through multiple pictures of the same cell. Figure 3.4 shows a timeseries of one cell over the course of 1 min. This type of movement was not observed for all dots, but only for a smaller population. This could support the hypothesis that most dots are in the gel-like state, while some still having a dynamic character.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-spd5-03.gif)

**Figure 3.4: Time series of dynamic liquid droplets.** MG1655 was transformed with pBAD33 containing SPD-5::sfGFP fusion protein and induced with 0.005% Arabinose. Panel show GFP fluorescence. SPD5::sfGFP form dynamic dots. Pictures were acquired every 5 sec for 1 min (scale bar 5 µm).

 Fluorescent recovery after photobleaching (FRAP) was perfomed on SPD-5::sfGFP fusion construct expressed in MG1655 as this showed some signs of dynamic droplets (Fig. 3.5). However, even after 30 min fluorescence is not recovered. It is worth noting that none of the photobleached dots showed dynamic characteristics.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-spd5-03-5.gif)

**Figure 3.5**: Time series of photobleaching of liquid droplets. MG1655 was transformed with pBAD33 containing SPD-5::sfGFP fusion protein. Panel show GFP fluorescence. SPD5::sfGFP fluorescent dots do not recover. Pictures were acquired every 30 sec for 30 min (scale bar 5 µm).

After having seen the ability of the SPD-5 protein to form liquid droplets *in vivo*, we could move on to test the tag-containing SPD-5 construct for its ability to form covalent bonds to catcher-containing mVenus and mTurquoise using fluorescent microscopy and western blot. These constructs were cloned into the same backbone as the microcompartments, pBbE6a. this allows for better comparison across different compartments. The mTurquoise and mVenus constructs are the same used for the microcompartments.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/plasmids-spd5-2.png)

**Aim**: To observe wether fluorescent "foci" are formed even after the fusion N- and C-terminally to the tags. To observe wether only successful catching of catcher-containing fluorescent proteins leads to fluoresence "foci".

**Experimental setup:** Strains are prepared in BL21 and are co-transformed with either pBbE6a containing SPD-5 or SPD-5 with spy- and snoop-tag on N- and C-term, respectively and pBbA2c containing either mVenus or mTurqouise. Strains were induced at OD: 0.6-0.8 and then incubated for 24h at 18°C. IPTG and Doxycyclin induces the expression of SPD-5 and mVenus/mTurquoise, respectively. Samples were induced with 100 µM IPTG and 25 ng/ml Doxycycline for mVenus and mTurquoise. Visualizing *via* fluorescent microscopy will allow us to observe SPD-5 based droplets only in the case where mVenus or mTurquise is catched and therefor colocalise inside the cell.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-spd5-04.png)

**Figure 3.6**: Fluorescent microscopy of cells expressing SPD-5 and mVenus. BL21 was co-transformed with either A: a plasmid with SPD-5 as well as a plasmid expressing mVenus or B: a plasmid with SPD-5 with spy- and snoop-tag on N- and C-term as well as a plasmid expressing mVenus. Bacteria expressing SPD-5 and mVenus form dots with strong fluorescent intensity for both SPD-5 constructs (scale bar 5 µm).Representive pictures of a single replicate.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-spd5-3-7.png)

**Figure 3.7: Fluorescent microscopy of cells expressing SPD-5 and mTurquoise.** BL21 was co-transformed with either A: a plasmid with SPD-5 as well as a plasmid expressing mTurquoise or B: a plasmid with SPD-5 with spy- and snoop-tag on N- and C-term as well as a plasmid expressing mTurquoise. Bacteria expressing SPD-5 and mTurquoise form dots with strong fluorescent intensity for both SPD-5 constructs (scale bar 5 µm).Representive pictures of a single replicate.

Figure 3.6 shows the expression of the cloned constructs in BL21 with fluorescent signal dispersed over the cytoplasm when mVenus is expressed alone and the occurrence of areas with strong intensity when it is expressed in the presence of SPD-5. In this case, we observe no visible difference for whether SPD-5 contains the spy- and snoop-tag or not. This suggests that the dots we are observing are either inclusion bodies or that the fluorescent protein is interacting with SPD-5 in a noncovalent manner. However, if these fluorescent dots were only due to aggregated proteins we would expect similar result when expressing another protein together with mVenus. The experiments done with mVenus and the T1 protein from the microcompartments (see Figure) does not show the same tendency and this suggests that these structures are related to the expression of SPD-5. Figure 3.7 shows very similar results for the co-expression with mTurquoise. In contrast to mVenus, the expression of mTurqoise is stronger and the "foci" are larger and occasionally form large structures covering the half of the cell. The high intensity dots are not moving or seemingly dynamic.

**MG1655**

**Aim:**As BL21 did not show same type of dynamic droplets observed with the fusion construct in MG1655, we also decided to use MG1655 for the same experimental setup to see if this would change the characteristics of the "foci". Besides, lower concentrations of IPTG were choosen to lower the risk of forming inclusion bodies.

**Experimental setup:** Strains are prepared in MG1655 and are co-transformed with either pBbE6a containing SPD-5 or SPD-5 with spy- and snoop-tag on N- and C-term, respectively and pBbA2c containing either mVenus or mTurqouise. Strains were induced at OD: 0.6-0.8 and then incubated for 24h at 18°C. IPTG and Doxycyclin induces the expression of SPD-5 and mVenus/mTurquoise, respectively. Samples were induced with 10 µM IPTG and 25 ng/ml Doxycycline for mVenus and 10 ng/ml for mTurquoise.

In contrast to the experiments for the SPD-5::sfGFP fusion construct in MG1655, the fluorescent "foci" are still stable and do not seem dynamic. As seen in Figure 3.8, the results are similar to those observed in BL21 and the formation of fluorescent dots is observed for both the construct of SPD-5 containing no tags. Meaning the fluorescent microscopy does not show a clear picture of whether the catching of mVenus worked as the observed dots could be due to general overproduction of proteins, unspecific binding to SPD5 etc. Therefore, western blot of these samples was prepared.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-spd5-05.png)

**Figure 3.8:** Fluorescent microscopy of cells expressing SPD-5 and mVenus. MG1655 was co-transformed with either A: a plasmid with SPD-5 as well as a plasmid expressing mVenus or B: a plasmid with SPD-5 with spy- and snoop-tag on N- and C-term as well as a plasmid expressing mVenus. Bacteria expressing SPD-5 and mVenus form dots with strong fluorescent intensity for both SPD-5 constructs (scale bar 5µm).

Figure 3.9 shows the western blot for the catching of mVenus. The expected size of mVenus with the spy-catcher is 37 kDa and the expected size when bound to SPD-5 is 167 kDa. In the case where a covalent bond between catcher and tag is created, a shift of mVenus band would be expected. The western blot shows a band at the right size of mVenus for the induced samples (lane 3, 5, 8, 10). In lane 10 a shift in the band for mVenus is detected at the expected size. This suggests that SPD-5 is capable of catching constructs with a spy-catcher.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-spd5-07.png)

**Figure 3.9**: Western blot of catching between SPD-5 and mVenus. MG1655 was co-transformed with either a plasmid with SPD-5 as well as a plasmid expressing mVenus (lane 2, 3, 4, 5) or a plasmid with SPD-5 with spy- and snoop-tag on N- and C-term as well as a plasmid expressing mVenus (lane 7, 8, 9, 10). There is a weak shift for the mVenus band when SPD-5 with spy- and snoop-tag is expressed.

Figure 3.10 shows the westernblot of the same samples as above, for SPD-5 and mTurquoise. The figure shows similar results to the previous western blot. In agreement with the observations for mVenus the transfer of the large catched construct (lane 10) shows a clearer band. It also shows that there are a band at about 60 kDa when mTurquoise is expressed in the strain containing SPD-5 with tags. These bands are not observed when mTurquise is expressed with SPD-5 without tags. These results show that the SPD-5 with spy-and snoop-tag is capable of catching the respective cargo proteins, which the SPD-5 alone is not capable of.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/anne/rs-spd5-08.png)

**Figure 3.10**: Western blot of catching between SPD-5 and mTurquoise. MG1655 was co-transformed with either a plasmid with SPD-5 as well as a plasmid expressing mTurquoise (lane 2, 3, 4, 5) or a plasmid with SPD-5 with spy- and snoop-tag on N- and C-term as well as a plasmid expressing mTurquoise (lane 7, 8, 9, 10). There is a shift for the mTurquoise band when SPD-5 with spy- and snoop-tag is expressed.

**Conclusion**

Based on these results we were able to show that SPD-5 fused to GFP formed fluorescent dots in MG1655 and in BL21 which we identify as some state of liquid droplets. We could show that after 24 h at 18°C it was possible to find single dynamic droplets changing position in the cell. However, as described previously [[3.1]](#re_ref3_1_2) the droplets transition over time to a gel-like state where the droplets localise to the poles and have little to no movement. In agreement, it was not possible to observe recovery of the fluorescent dots after photobleaching possibly due to the long induction time.

We also created a construct with SPD-5 containing spy and snoop-tag allowing catching *via* spy or snoop catcher. We showed that these strains still form fluorescents dots when expressed in BL21 and MG1655. However, in agreement with the fusion construct these droplets do not seem to be in the dynamic state but rather in a gel-like state. We could confirm *via* western blot that even though we observe fluorescent dots when SPD-5 is expressed without tags together with catcher containing mVenus or mTurquoise, they do not catch either mVenus or mTurquoise.

**Future perspectives**

Our project seeks to compare types of compartents for co-localizing proteins. From this view, there would be several considerations to make when choosing to work with Liquid droplets. First of all, as is generally known, the formation of liquid droplets is sensitive to protein concentration and induction time [[3.4]](#re_ref3_4). As the gel-like state is not as dynamic it might be less suitable for increasing product yield due to less exchange with the cytoplasma. Testing different induction conditions for a liquid droplet forming protein of interest would be necessary. Along the same lines, having the possibility to further regulate the formation of droplets and possibly avoid them turning to the gel-like state could be beneficial.

## Optimisation with ncAAs

**Key findings:**

* We have shown not only that ncAAs can be incorporated into Wiffleball, but also that they can, *via* crosslinking, give new properties to the shell.
* We created multiple amber stop codon mutants of sfGFP and verified the incorporation of two ncAAs at these positions
We released these variants as part collection to the registry. It can be used by future iGEM teams to test the incorporation of other ncAAs

The different mutations of the BMC-T1-spyt-snpt-6xHis protein were not randomly selected. We selected potential non-conserved amino acids using the consurf database. We followed the generally accepted view that the less conserved an amino acid is, the less relevant it is for the correct folding of the protein. The remaining non-conserved amino acids and their positions were considered and only amino acids that had a similar size and shape compared to the ncAAs were selected for site directed mutagenesis at their position.

This resulted in the mutations: BMC-T1-spyt-snpt-6xHis F8, T35, R78 and Y96.

The same applies to the mutations introduced into sfGFP.

## ncAAs in GFP

### **Detecting the incorporation of non-canonical amino acids by flow cytometry**

Besides the well-known 20 canonical amino acids, there is a variety of non-canonical amino acids (ncAAs) which can be used to further modify proteins. One way of incorporating ncAAs is *via* the amber stop codon suppression technology.

An aminoacyl-tRNA synthetase (aaRS) and a tRNA (orthogonal) derived from other species must be introduced into the organism. The tRNA must not be recognised by the aaRSs of the organism, the same applies to the introduced aaRS and tRNAs of the cell. In addition, the new tRNA must introduce the ncAA *via* a codon that does not encode any of the 20 natural amino acids, and the ncAA must be taken up by the cell. The requirements described above are fulfilled by E. coli in combination with the evolved tyrosyl-tRNA/synthetase pair of Methanococcus jannaschii. Amber stop codon (UAG) suppression technology is used for this purpose, whereby the codon is recognised by the tRNA, and the loaded ncAA is incorporated.

**Testing the incorporation of** **4-Azido-L-phenylalanine (*****p*****-AzF) into sfGFP**

**Aim:** Indirectly test the incorporation of *p*-AzF into sfGFP measuring fluorescence using flow cytometry under different induction levels

**Experimental design:**
*E. coli* BL21 (DE3) cells were co-transformed with the plasmid coding for the pAzF synthetase and the corresponding tRNA, and the plasmid coding for the sfGFP Y74 mutant (pTrc99a\_sfGFP Y74; [http://parts.igem.org/Part:BBa\_K4229023](http://parts.igem.org/Part%3ABBa_K4229023)).

Colonies from the co-transformation were  grown overnight at 37°C in LB medium supplemented with ampicillin and chloramphenicol. Overnight cultures were diluted and grown to OD600 of 0.4 at 30°C before induction. Samples were induced with different arabinose concentrations as indicated in Figure 4.1 1 mM *p-*AzF was added into the medium, unless differently indicated (for instance, for the control, in which no ncAA was included). One hour after arabinose induction (which triggers expression of the tRNA synthetase), the samples were induced with 500 µM IPTG to induce sfGFP and growth was continued for additional 3 h at 30°C.

The incorporation of the ncAA was indirectly measured by fluorescence, which was quantified by flow cytometry with the CyAn ADP Analyzer from Beckman Coulter. Samples were prepared by mixing 10 µL of the liquid bacterial cultures with 990 µL Dulbecco's Balanced Salt Solution

(DPBS).

**Results:**

As showcased in Figure 4.1, different levels of arabinose do not seem to significantly affect the median fluorescence intensity (MFI) of the cells expressing the sfGFP mutant or wild-type. This is of course expected for WT sfGFP given that arabinose is required for the expression of the orthogonal tRNA synthetase, which is not needed for the WT protein. The MFI of the cells expressing the mutant is consistently lower than that of the cells expressing wild-type sfGFP. We were not surprised by this result, considering that we do not expect 100% efficiency of ncAA incorporation. Nonetheless, even at very low arabinose levels, incorporation of *p-*AzF is occurring. Interestingly, fluorescence can be detected even without induction with arabinose, but in the presence of *p-*AzF. We speculate this is due to the leakiness of the promoter, which leads to the expression of the synthetase even without arabinose. It moreover suggests that very low amounts of synthetase are sufficient, which is in line with the results obtained with increasing amounts of arabinose (Fig. 4.1).

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/araconc.png)

**Figure 4.1:** Bar graph showing the Median Fluorescence Intensity (MFI) of E.coli BL21 (DE3) cells transformed with pEVOL-pAzF and pTrc99a coding for the indicated GFP variant induced with the indicated arabinose concentrations. GFP fluorescence was measured by flow cytometry. 500 µM IPTG were used to induce sfGFP expression. 1 mM p-AzF were added to all samples except for the sample labelled “N.C.”, which did not receive p-AzF. This sample was also not induced at all (no arabinose, no IPTG).

### **Influence of ncAAs and the synthetases on wild-type sfGFP fluorescence**

**Aim:** Explore the impact of the ncAA and/or the expression of the aminoacyl tRNA synthetase on the median fluorescence intensity of wild-type sfGFP.

**Experimental setup:** *E. coli* BL21(DE3) were co-transformed with the plasmid coding for either the pAzF or the pBpF synthetase, and the plasmid coding for wild-type sfGFP (pTrc99a-sfGFP).

A single colony from each co-transformation was grown overnight at 37°C in LB medium supplemented with ampicillin and chloramphenicol. Overnight cultures were diluted and grown to OD600 of 0.4 at 37°C before induction. Samples were induced with 1mM arabinose (to induce the expression of the synthetase). 1 mM *p-*AzF or 0.2 mM *p-*BpF were added to all samples except in the controls, where the ncAA were omitted. One hour after arabinose induction the samples were induced with 200 µM IPTG (to allow expression of sfGFP) and growth was continued for additional 2.5 h at 37°C

The incorporation of the ncAA was indirectly measured by fluorescence, which was quantified by flow cytometry with the CyAn ADP Analyzer from Beckman Coulter. Samples were prepared by mixing 10 µL of the liquid bacterial cultures with 990 µL DPBS.

**Results:**

We expected no difference in the GFP fluorescence across all the samples, given that WT sfGFP does not rely on the orthogonal tRNA/tRNA synthetase pair and does not incorporate the ncAA. We maximally expected to see lower fluorescence in cells expressing the synthetase, because of the burden of producing an additional heterologous protein. The results we obtained were quite surprising and puzzling (Figure 4.2). Since we performed this experiment only once, we tend to be cautious in interpreting the data. While doing the experiments, we indeed realised that the GFP fluorescence varied a lot from day to day and we could not always pin down the reason for it.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/impactofncaaorrs.png)

**Figure 4.2:** Bar graph showing the Median Fluorescence Intensity (MFI) of BL21(DE3) cells transformed with pEVOL-pAzF (pAzFRS), pEVOL-pBpF (pBpf RS) or empty pBAD33 (pBAD33). GFP fluorescence was measured by flow cytometry. 500 µM IPTG were used to induce sfGFP expression. 1 mM pAzF or 0.2 mM p-BpF were added to all samples labelled with the “+”

### **Testing different positions within sfGFP for the incorporation of ncAAs**

**Aim:** Design a toolbox for the easy quantification of the efficiency of ncAA incorporation *via* the amber stop codon suppression technology.

**Experimental setup:**

We introduced the amber stop codon at positions G4, F8, W57, Y74, Y151 and D216 in sfGFP by site directed mutagenesis.  *E. coli* BL21(DE3) were co-transformed with the following combinations of plasmids:

* pEVOL-pBpF and pTrc99a-sfGFP WT
* pEVOL-pBpF and pTrc99a-sfGFP G4
* pEVOL-pBpF and pTrc99a-sfGFP F8
* pEVOL-pBpF and pTrc99a-sfGFP W57
* pEVOL-pBpF and pTrc99a-sfGFP Y74

* pEVOL-pBpF and pTrc99a-sfGFP Y151
* pEVOL-pBpF and pTrc99a-sfGFP D216
* pBAD33 and pTrc99a-sfGFP WT
* pBAD33 and pTrc99a-sfGFP G4
* pBAD33 and pTrc99a-sfGFP F8

* pBAD33 and pTrc99a-sfGFP W57
* pBAD33 and pTrc99a-sfGFP Y74
* pBAD33 and pTrc99a-sfGFP Y151
* pBAD33 and pTrc99a-sfGFP D216
* pBAD33 and pTrc99a

A single colony from each co-transformation was grown overnight at 37°C in LB medium supplemented with ampicillin and chloramphenicol. Overnight cultures were diluted and grown to OD600 of 0.4 at 37°C before induction. Samples were induced with 1mM arabinose and 0.2 mM of *p-*BpF were added in all samples except in the control. One hour after arabinose induction the samples were induced with 200 µM IPTG and growth was continued for additional 2.5 h at 37°C

The incorporation of the ncAA was indirectly measured by fluorescence, which was quantified by flow cytometry with the CyAn ADP Analyzer from Beckman Coulter. Samples were prepared by mixing 10 µL of the liquid bacterial cultures with 990 µL DPBS.

**Results:**

Interestingly, not all mutations exhibited fluorescence in the presence of *p-*BpF and the corresponding synthetase (Figure 4.3). This suggests that the amino acid context plays a role in facilitating or inhibiting the incorporation of a ncAA.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/allmutationserr.png)

**Figure 4.3:**Bar graph showing the Median Fluorescence Intensity (MFI) of BL21(DE3) cells transformed with pEVOL-pBpF and the indicated sfGFP variants. GFP fluorescence was measured by flow cytometry. 200 µM IPTG were used to induce sfGFP expression. 0.2 mM p-BpF were added to all samples labelled with the “+”. Blue indicates that no synthetase was present (-) but the medium contained 0.2 mM p-BpF (+), orange stands for the presence of both the synthetase (+) and 0.2 mM p-BpF (+), and grey indicates the presence of the synthetase (+) and the absence of p-BpF (-). Data are means ± standard deviation of two independent experiments.

### **Photo-crosslinking of bacterial microcompartment subunits with p-benzoyl-l-phenylalanine**

P-benzoyl-l-phenylalanine (*p*-BpA or *p*-Bpf) is a non-canonical amino acid with a benzophenon residue and commonly used for photochemical crosslinking [[4.1]](#re_ref4_1). When irradiated with UV light it can form covalent bonds with nearby C-H bonds. The reaction mechanism for photo-crosslinking with *p-*Bpf is shown in the figure below.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/bpbfreactionmech.png)

**Figure 4.4: Irradiation of p-Bpf at 365 nm:** If p-Bpf is irradiated with a wavelength of 365 nm, a diradical species is generated. If another molecule, with an activated C-H group, approaches, a radical also occurs at this molecule, since the H binds to one of the two previous radicals. Subsequently, a radical recombination occurs and the two molecules are linked, adapted from [[4.2]](#re_ref4_2).

For the following experiments *p-*Bpf was incorporated *via* amber stop codon suppression technology with an orthogonal translation system consisting of an aminoacyl tRNA synthetase (from Addgene, pEVOL-pBpF (plasmid #31190)) and the corresponding tRNA.

The amber stop codons were introduced with the help of site directed mutagenesis at the positions F8, T35, R78 and Y96.

**Aim:** Covalently crosslinking the building blocks of microcompartments can significantly impact their stability. With this experiment we want to show that *in vivo* crosslinking of the subunits of our bacterial microcompartments is possible by utilizing non-canonical amino acids (ncAAs).

**Experimental Setup:**  *E.coli* BL21 (DE3) were co-transformed with the following combinations of plasmids:

* pEVOL-pBpF and pHT1\_spyt\_snptT2T3 F8
* pEVOL-pBpF and pHT1\_spyt\_snptT2T3 T35
* pEVOL-pBpF and pHT1\_spyt\_snptT2T3 R78
* pEVOL-pBpF and pHT1\_spyt\_snptT2T3 Y96
* pBAD33 and pHT1\_spyt\_snptT2T3 WT
* pBAD33 and pHT1\_spyt\_snptT2T3 T35

A single colony from each co-transformation was grown overnight at 37°C in LB medium supplemented with ampicillin and chloramphenicol. The first five overnight cultures were transferred into fresh Lb medium supplemented with 0.2 mM unnatural amino acid, *p-*Bpf and grown to OD600 of 0.4 at 37°C before induction with 1mM arabinose. One and a half hours after arabinose induction the samples were induced with 200 µM IPTG and growth was continued 28 h at 18°C. The last construct (pBAD33 and pHT1\_spyt\_snptT2T3 T35) was not induced and no ncAA was added, it is the negative control.

To crosslink the proteins, 6 ml of the medium per batch was centrifuged at 5000 g, and the supernatant was discarded. 3.6 ml of PBS was used to resuspend the cells, then these were centrifuged at 3000 g. The supernatant was discarded again, and the pellet was resuspended in 3 ml PBS. Samples were placed in 47 mm petri dishes. The samples were irradiated with a UVP crosslinker from analytikjena and 100 μl per sample were collected after each irradiation step. Samples were then centrifuged and resuspended with Lämmli buffer and heated to 95 °C for 5 min. Then they were separated using an SDS gel and a Western blot was made.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/a8-a35.png)
![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/a78-a96.png)

**Firgure 4.5: The different mutations are represented by red spheres in the T1 protein of the HO shell for spatial viewing.** The three subunits of the pseudohexamer were coloured different. (A) Mutation F8 in each of the three subunits of the pseudohexamer. (B) Mutation T35. (C) Mutation R78. (D) Mutation Y96. Images were generated using PyMOL [[4.3]](#re_ref4_3).

In Figure 4.5, it can be seen that no cross-linking is expected for F8 and R78 because the mutations are located in the centers of the three subunits, respectively, and thus *p-*Bpf cannot come into spatial proximity with other proteins and cannot form a covalent bond. For the T35 mutation, based on the figure, it can be assumed that a crosslink to a neighboring T1 protein, is possible. The same is true for Y96, but here the formation of a bond also to other proteins such as the H-Protein is likely, since *p-*Bpf is located on the outside.

**Results:**

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/a96-07-10.png)

**Figure 4.6: Western blot of BMC-T1-spyt-snpt-6xHis Y96 expression.**  E. coli (BL21) was co-transformed with pEVOL-pBpF and pHT1\_spyt\_snptT2T3 Y96. Arabinose and IPTG induced the expression of the aminoacyl-tRNA synthetase and the proteins of the plasmid pHT1\_spyt\_snptT2T3, p-Bpf was added. BMC-T1-spyt-snpt-6xHis are detected using anti-his antibody, loading control is the RNA polymerase ß-subunit.

In the unirradiated sample (Fig. 4.6, left), it can be clearly seen that only the bands of the *BMC-T**1-spyt-snpt-6xHis* protein and *the RNA polymerase ß-subunit* are visible. For the other samples, one can clearly see bands at other heights. Especially the bands between 72 and 100 kDa stand out. However, this does not indicate a connection to H-proteins as initially assumed but suggests connections to another *BMC-T**1-spyt-snpt-6xHis* or a protein of similar size.

Due to the particularly strong bands in rows three, four and five, the parameters used were classified as suitable for crosslinking. Therefore, for the following two Western blots, these samples, among others, were selected for the other mutations.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/gel1-0910.png)

**Figure 4.7: Western blot of BMC-T1-spyt-snpt-6xHis F8, T35 and controls pHT1\_spyt\_snptT2T3 WT and pHT1\_spyt\_snptT2T3 T35 uninduced.** The table shows which samples were loaded and with which intensity they were irradiated for how long. Arabinose and IPTG induced the expression of aminoacyl-tRNA synthetase and proteins of plasmid pHT1\_spyt\_snptT2T3, p-Bpf was added. This applies to all samples except the one in row two, which was not induced and no ncAA was added. BMC-T1-spyt-snpt-6xHis are detected using anti-his antibody, loading control is the RNA polymerase ß-subunit.

Figure 4.7 shows that BMC-T1-spyt-snpt-6xHis F8 does not show crosslinking to other proteins as expected, however this is also the case for T35, previous experiments came to the same conclusion.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/gel2-0910.png)

**Figure 4.8: Western blot of BMC-T1-spyt-snpt-6xHis R78, Y96 and controls pHT1\_spyt\_snptT2T3 WT and pHT1\_spyt\_snptT2T3 T35 uninduced.**  The table shows which samples were loaded and with which intensity they were irradiated for how long. Arabinose and IPTG induced the expression of aminoacyl-tRNA synthetase and proteins of plasmid pHT1\_spyt\_snptT2T3, p-Bpf was added. This applies to all samples except the one in row two, which was not induced and no ncAA was added. BMC-T1-spyt-snpt-6xHis are detected using anti-his antibody, loading control is the RNA polymerase ß-subunit.

Figure 4.8 shows that BMC-T1-spyt-snpt-6xHis R78 is not cross-linked with other proteins as expected. For BMC-T1-spyt-snpt-6xHis Y96, the same samples as in Figure 4.7 were loaded and cross-linking could again be detected between 72 and 100 kDa.

The first Western blot (Fig. 4.6) was performed on the same day as the cross linking. Interestingly, this one shows stronger bands than the western blots two and three (Fig. 4.7 and 4.8),especially for cross linking. For these, the exact same amounts were loaded, but the samples were frozen overnight and thawed the next day for the blots. The fact that the bands produced less intense signals after freezing, under otherwise exactly the same conditions, was observed several times throughout the project.

## NcAA in wiffleballs

**Short Abstract about achievements**

Our goal is to introduce non-canonical amino acids (ncAAs) at specific positions into wiffleballs (bacterial microcompartment shells derived from Haliangium ochraceum also referred to as BMCs) to allow cross-linking with other proteins at these positions (pBpF, p-benzoyl-l-phenylalanine) or to introduce amino acids with azido groups as side chains for possible click chemistry reactions (pAzF, p-azido-l-phenylalanine). We used rational design by first looking at the crystal structure with PyMOL while colouring the amino acids based on their conservation using the ConSurf database and looking for positions that were not highly conserved but close to other compartment components. We decided to look at the BMC-T1-F8TAG, BMC-T1-T35TAG, BMC-T1-R78TAG and BMC-T1-Y96TAG mutations. To detect the incorporation of the amino acid, we tested different conditions and detected them with Western blots using an α-HisTag antibody. Finally, to establish that these mutant wiffleball proteins can still be caught by the SpyCatcher and SnoopCatcher systems associated with our pathway enzymes, we tested all systems together under different conditions and detected them with an α-HisTag as well as α-Catcher antibody.

**Results:**
In order for the incorporation of the ncAA to succeed, an amber stop codon (TAG) was inserted at amino acid position 35 (Fig. 4.5A) in the DNA sequence, where a threonine is normally located. This construct (BMC-T1-T35TAG) was then transformed with aminoacyl tRNA synthetase (aaRS) and the complementary tRNA.
**Aim:**
We looked at the BMC-T1-T35TAG mutant in *E. coli* BL21 (DE3) and performed various tests with it to characterise the other mutations more quickly. We want to show the incorporation of the ncAA into the protein.
**Experimental design:**
Matching controls were also transformed so that the incorporation can be attributed to the addition of the ncAA, the expression of the aaRS/tRNA and the expression of the BMC-T1-T35TAG mutant. As controls, we transformed "pBAD + pHT1spysnpT2T3" as the pHT1spysnpT2T3 plasmid contains all the constructs required to produce the complete wiffleball structure (BMC C). The BMC-T1-T35TAG mutant was created through site directed mutagenesis of the pHT1spysnpT2T3 construct to still be able to benefit from the structure of a whole wiffleball instead of the minimal wiffleball.
Transformed controls in *E. coli* BL21 (DE3):

* pBAD + pTrc99a
* pBAD + pHT1spysnpT2T3
* pBAD + BMC-T1-T35TAG
* pEVOL-pAzFRS + pHT1spysnpT2T3
* pEVOL-pAzFRS + BMC-T1-T35TAG

For cells treated with pAzF, 1 mM pAzF was added into the medium before growing the cells. The cells were grown until an OD of 0.4, were then treated with 1 mM arabinose and grew for two more hours at 37 °C. After, the cells were treated with 100 µM of IPTG, grew for 5 more hours at 30 °C and were harvested by centrifugation. The samples were diluted with Laemmli buffer depending on the OD600 of the cell culture, and then heated for 5 minutes at 95°C. After, the samples were loaded on an SDS-PAGE, running for 2.5 hours with constant 0.01 mA per gel. Then, the samples were wet blotted for 2 hours at 0.38 mA. An anti-6xHisTag antibody (1:1000) as well as an anti RNA polymerase antibody (1:1000) in combination with an anti-mouse-HRP secondary antibody (1:10000) were used for detection. The anti-6xHisTag antibody should only detect the BMC-T1 protein due to the N-terminally added 6xHis-Tag.

**Results:** The Western blot (Fig. 4.10) shows a band at the level of slightly above 35 kDa for "pBAD + pHT1spysnpT2T3" induced with IPTG, "pEVOL-pAzFRS + pHT1spysnpT2T3" induced with IPTG only and IPTG plus arabinose, and for "pEVOL-pAzFRS BMC-T1-T35TAG", upon addition of 1 mM pAzF and induction by IPTG and addition of 1 mM pAzF and induction by IPTG + arabinose.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/image-1.png)

**Figure 4.10: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 35 of the BMC-T1 protein.** The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 5 hours before harvesting by centrifugation. The PageRuler™ Prestained Protein Ladder, 10 to 180 kDa from ThermoFisher Scientific was used. For detection, an anti 6xHisTag (1:1000) as well as an anti RNA polymerase (1:1000) were used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, Ara – arabinose, pAzF – p-azido-l-phenylalanine.

The bands seen at 35 kDa is assumed to be our proteins of interest, as samples possessing a plasmid with the BMC-T1 gene and being induced with IPTG should induce our BMC-T1 protein. The BMC-T1 was detected by ECL due to its N-terminal 6xHisTag antibody.
**Conclusion**Interestingly, we thought that BMC-T1 should be found at a size of 29 kDa, but the band runs at 35 kDa. However, there is no gene on the BMC plasmid that has a 6xHisTag and is 35 kDa in size. The same effect also occurs within other membranes.
The aaRS for pAzF does not appear to affect the expression of the BMC-T1 protein. For our sample, which has the aaRS, tRNA as well as the BMC-T1-T35TAG plasmid, a clear band is seen but not in the absence of pAzF despite induction by IPTG and arabinose. Therefore, we can assume that specifically pAzF is incorporated into this protein. Interestingly, a band is seen in the "pEVOL-pAzF BMC-T1-T35TAG" sample where 1 mM pAzF was added to the medium and induced with only 100 µM IPTG. However, this band can be explained due to synthetase having two variants of the synthetase, one with a mid-strength *glnS* promoter next to the synthetase and the other with a lac promoter induced by IPTG.
After the expression of BMC-T1-T35TAG worked well with the incorporation of pAzF, we also tested the three other mutations (Fig. 4.5 B-C) with pAzF (Figure 4.11 + 4.12) and all four mutations with pBpF (Figure 4.13 + 4.14) as well. As we figured out by previous experiments with the synthetic full wiffleball construct (BMC C, pHT1spysnpT2T3), we increased the growth time after ITPG induction to 24 hours at 18°C to maximise the production of the protein.
**Experimental design:** The following transformations of *E. coli* BL21 (DE3) cells were used:

* BAD + pTrc99a
* pBAD + pHT1spysnpT2T3
* pBAD + BMC-T1-T35TAG
* pEVOL-pAzFRS + pHT1spysnpT2T3
* pEVOL-pAzFRS + BMC-T1-F8TAG
* pEVOL-pAzFRS + BMC-T1-T35TAG
* pEVOL-pAzFRS + BMC-T1-R78TAG
* pEVOL-pAzFRS + BMC-T1-F96TAG
* pEVOL-pBpFRS + BMC-T1-F8TAG
* pEVOL-pBpFRS + BMC-T1-T35TAG
* pEVOL-pBpFRS + BMC-T1-R78TAG
* pEVOL-pBpFRS + BMC-T1-F96TAG

For cells treated with pAzF, 1 mM pAzF was added into the medium before growing the cells. The cells were grown until an OD of 0.4, were then treated with 1 mM arabinose and grew for two more hours at 37 °C. After, the cells were treated with 100 µM of IPTG, grew for 5 more hours at 30 °C and were harvested by centrifugation. The samples were diluted with Laemmli buffer depending on the OD600 of the cell culture, and then heated for 5 minutes at 95°C. After, the samples were loaded on an SDS-PAGE, running for 2.5 hours with constant 0.01 mA per gel. Then, the samples were wet blotted for 2 hours at 0.38 mA. An anti-6xHisTag antibody (1:1000) as well as an anti RNA polymerase antibody (1:1000) in combination with an anti-mouse-HRP secondary antibody (1:10000) were used for detection. The anti-6xHisTag antibody should only detect the BMC-T1 protein due to the N-terminally added 6xHis-Tag.
**Results:**
We get the same results for BMC-T1-T35TAG (Fig. 4.11), -R78TAG, and -Y96TAG (Fig. 4.12) when incorporating pAzF, as well as for BMC-T1-F8TAG, -T35TAG (Fig. 4.13), -R78TAG, and -Y96TAG (Fig. 4.14) when incorporating pBpF.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/image-2.png)

***Figure 4.11: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 8 and 35 of the BMC-T1 protein.*** *The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The PageRuler™ Prestained Protein Ladder, 10 to 250 kDa from ThermoFisher Scientific was used. For detection, an anti 6xHisTag (1:1000) as well as an anti RNA polymerase (1:1000) antibody were used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, pAzF – p-azido-l-phenylalanine.*

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/image-3.png)

***Figure 4.12: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 78 and 96 of the BMC-T1 protein.***  *The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The PageRuler™ Prestained Protein Ladder, 10 to 250 kDa from ThermoFisher Scientific was used. For detection, an anti 6xHisTag (1:1000) as well as an anti RNA polymerase (1:1000) antibody were used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, pAzF – p-azido-l-phenylalanine.*

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/image-4.png)

***Figure 4.13: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pBpF at position 8 and 35 of the BMC-T1 protein.*** *The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The PageRuler™ Prestained Protein Ladder, 10 to 250 kDa from ThermoFisher Scientific was used. For detection, an anti 6xHisTag (1:1000) as well as an anti RNA polymerase (1:1000) antibody were used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, pBpF – p-benzoyl-l-phenylalanine.*

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/image-5.png)

***Figure 4.13: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pBpF at position 78 and 96 of the BMC-T1 protein.***  *The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The PageRuler™ Prestained Protein Ladder, 10 to 250 kDa from ThermoFisher Scientific was used. For detection, an anti 6xHisTag (1:1000) as well as an anti RNA polymerase (1:1000) antibody were used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, pBpF – p-benzoyl-l-phenylalanine.*

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/ncaa/image-6.png)

***Figure 4.14: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pBpF at position 78 and 96 of the BMC-T1 protein.***  *The cells grew until OD 0.4, following the addition of arabinose and a growth period of 2 h. After the 2 h the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used as ladder. To purify the protein, the His-Spin Protein Miniprep from Zymo Research was used.*

**Discussion:** As can be seen in figure 3, there were problems detecting the production of BMC-T1-F8TAG with pAzF, due to an unnoticed air bubble remaining in the SDS gel during polymerisation. However, we can provide further evidence of this protein in figure 7, as the mutants with incorporated pAzF were purified using a HisTag column and loaded onto an SDS-PAGE, to confirm the working purification for further analysation via mass spectrometry.

![Figure 4.15: SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 8, 35, 78 and 96 of the BMC-T1 protein. ](https://static.igem.wiki/teams/4229/wiki/pictures/re-ncaa-415.png)

***Figure 4.15: SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 8, 35, 78 and 96 of the BMC-T1 protein.**  The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used as ladder. To purify the protein, the His-Spin Protein Miniprep from Zymo Research was used.*

The primary band (induced pHT1spysnpT2T3) in figure 4.15 was loaded as a positive control to see whether the band where the protein has been overexpressed corresponds to the band of the purified samples. The purified proteins run at ~29 kDa, despite being detected at 35 kDa on the western blots. There is no other construct containing an HisTag.
**Aim:** After we found out that the incorporation of the non-canonical amino acids we use works with every mutation, it was of course interesting to transform the plasmid containing the indigo enzymes or an empty plasmid with these constructs.
Before looking at the mutants, we expressed the non-mutant full wiffleball along with the indigo enzymes (Fig. 4.16). When only the BMC is transformed and expressed, a band at 35 kDa is evident, as has been detected in previous Western blots.

**Experimental design:**  The following transformations were inoculated:

* pTrc99a + pCDFDUET-1
* pTrc99a + sTAF sXTB
* pHT1spysnpT2T3 + pCDFDUET-1
* pHT1spysnpT2T3 + sTAF sXTB

The cells were grown until an OD of 0.4 and then treated with 200 µM of IPTG, grew for 24 hours at 18°C and were harvested by centrifugation. The samples were diluted with Laemmli buffer depending on the OD600 of the cell culture, and then heated for 5 minutes at 95°C. After, the samples were loaded on an SDS-PAGE, running for 2.5 hours with constant 0.01 mA per gel. Then, the samples were wet blotted for 2 hours at 0.38 mA. An anti-6xHisTag antibody (1:1000) as well as an anti RNA polymerase antibody (1:1000) in combination with an anti-mouse-HRP secondary antibody (1:10000) were used for detection. The anti-6xHisTag antibody should only detect the BMC-T1 protein due to the N-terminally added 6xHis-Tag.
**Results:** When only the full wiffleball is expressed together with the indigo enzymes, a band at 100 kDa is detected, suggesting that our enzymes are caught.
![Figure 4.16: Western blot of an SDS-PAGE with whole cell lysis confirming the catching of Indigo enzymes by the BMC-T1 protein.](https://static.igem.wiki/teams/4229/wiki/pictures/re-ncaa-416.png)

***Figure 4.16: Western blot of an SDS-PAGE with whole cell lysis confirming the catching of Indigo enzymes by the BMC-T1 protein.** The cells grew until OD600 0.4, following the addition of 200 µM IPTG and grew 24 hours before harvesting by centrifugation. The PageRuler™ Prestained Protein Ladder, 10 to 250 kDa from ThermoFisher Scientific was used. For detection, an anti 6xHisTag (1:1000) as well as an anti RNA polymerase (1:1000) antibody were used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside.*

**Aim:** We then decided to focus on the BMC-T1-T35TAG mutant and therefore have four different samples to test: mutant with pAzFRS or pBpFRS and empty indigo backbone (pCDFDUET-1) as well as mutant with pAzFRS or pBpFRS and the genes for the indigo enzymes (sTAF sXTB). We want to show the incorporation of the ncAAs as well as the catching of the enzymes simultaneously.
**Experimental design:** The following transformations were inoculated:

* pTrc99a + pBAD + pCDFDUET-1
* pTrc99a + pBAD + sTAF sXTB
* pHT1spysnpT2T3 + pBAD + pCDFDUET-1
* pHT1spysnpT2T3 + pBAD + sTAF sXTB
* BMC-T1-T35TAG + pBAD + sTAF sXTB
* BMC-T1-T35TAG + pEVOL-pAzFRS + pCDFDUET-1
* BMC-T1-T35TAG + pEVOL-pAzFRS + sTAF sXTB
* BMC-T1-T35TAG + pEVOL-pBpFRS + pCDFDUET-1
* BMC-T1-T35TAG + pEVOL-pBpFRS + sTAF sXTB

For cells treated with pAzF, 1 mM pAzF was added into the medium before growing the cells. The cells were grown until an OD of 0.4, were then treated with 1 mM arabinose and grew for two more hours at 37 °C. After, the cells were treated with 200 µM of IPTG, grew for 24 more hours at 18 °C and were harvested by centrifugation. The samples were diluted with Laemmli buffer depending on the OD600 of the cell culture, and then heated for 5 minutes at 95°C. After, the samples were loaded on an SDS-PAGE, running for 2.5 hours with constant 0.01 mA per gel. Then, the samples were wet blotted for 2 hours at 0.38 mA. An anti-6xHisTag antibody (1:1000) as well as an anti RNA polymerase antibody (1:1000) in combination with an anti-mouse-HRP secondary antibody (1:10000) were used for detection. The anti-6xHisTag antibody should only detect the BMC-T1 protein due to the N-terminally added 6xHis-Tag.
**Results:**
First, we decided to detect the 6xHisTag to see if the band would shift if the indigo enzymes were caught. We loaded controls for each blot to better prove our assumptions (Fig. 4.17-4.20). We get a band at the level of 35 kDa when only the full wiffleball is expressed. However, if the wiffleball is expressed together with the indigo enzymes, we still see a band at 35 kDa, but also at 100 kDa. If only the indigo enzymes are expressed, no band is seen, therefore, the 6xHisTag antibody has no interaction with the indigo enzymes.

![Figure 4.17: Western blot of an SDS-PAGE with whole cell lysis confirming the catching of Indigo enzymes by the BMC-T1 protein.](https://static.igem.wiki/teams/4229/wiki/pictures/re-ncaa-417.png)

***Figure 4.17: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pAzFRS, and pCDFDUET-1.** The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used as ladder. For detection, an α-6xHisTag antibody (1:1000) was used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, pAzF – p-azido-l-phenylalanine.*

![Figure 4.18: Western blot of an SDS-PAGE with whole cell lysis confirming the catching of Indigo enzymes by the BMC-T1 protein.](https://static.igem.wiki/teams/4229/wiki/pictures/re-ncaa-418.png)

***Figure 4.18: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pAzFRS, and sTAF sXTB.** The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used as ladder. For detection, an α-6xHisTag antibody (1:1000) was used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, pAzF – p-azido-l-phenylalanine.*

![Figure 4.19: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pBpF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pBpFRS, and pCDFDUET-1. ](https://static.igem.wiki/teams/4229/wiki/pictures/re-ncaa-419.png)

***Figure 4.19: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pBpF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pBpFRS, and pCDFDUET-1.** TThe cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used as ladder. For detection, an α-6xHisTag antibody (1:1000) was used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, pBpF – p-benzoyl-l-phenylalanine.*

![Figure 4.20: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pBpF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pBpFRS, and sTAF sXTB.  ](https://static.igem.wiki/teams/4229/wiki/pictures/re-ncaa-420.png)

***Figure 4.20: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pBpF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pBpFRS, and sTAF sXTB.** The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used as ladder. For detection, an α-6xHisTag antibody (1:1000) was used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, pBpF – p-benzoyl-l-phenylalanine.*

If the mutant BMC-T1 proteins are formed with pAzF or pBpF and simultaneously transformed with the pCDFDUET-1 plasmid (Fig. 4.17 + 4.19), no shift is seen, and it looks like the previous Western blots. However, when the mutants are transformed and expressed with sTAF sXTB, a clear band is seen at 100 kDa, which can also be seen for the positive control when the unmutated BMC and sTAF are transformed and expressed (Fig. 4.19 + 4.20).
**Conclusion**
The incorporation of the ncAAs as well as the catching was successful. In both variants (pAzF and pBpF) it can be seen that two bands exist (35 kDa and 100 kDa). Accordingly, more BMC-T1 could be produced than is necessary to capture all enzymes.
**Aim:** After detecting the membranes with the 6xHisTag antibody, we wanted to detect the same membranes with a catcher antibody, which should detect the catchers of the indigo enzymes and not BMC-T1.
**Experimental design:**
For this purpose, it is important to inactivate horse radish peroxidase (HRP) after the first detection so that it does not give a background signal during the next detection and thus distort the results. Sodium azide is also mixed into our primary antibodies so that the HRP can be inactivated in precisely such cases. After incubation with the catcher antibody and before incubation with the secondary antibody, the membranes were detected again via ECL to measure any remaining signal (Fig. 4.21). The membranes were detected for 2 minutes each and no signal is measured, therefore the catcher antibody can be detected reliably.

![Figure 4.21: Western blot of an SDS-PAGE with whole cell lysis after decorating the membranes with an α-6xHisTag antibody and α-mouse secondary antibody via ECL and then incubating with an α-Catcher antibody and detecting via ECL](https://static.igem.wiki/teams/4229/wiki/pictures/re-ncaa-421.png)

***Figure 4.21: Western blot of an SDS-PAGE with whole cell lysis after decorating the membranes with an α-6xHisTag antibody and α-mouse secondary antibody via ECL and then incubating with an α-Catcher antibody and detecting via ECL** After the membranes have been decorated and detected with anti-6xHisTag, the same membranes should also be detected and decorated with an anti-Catcher antibody. After incubation with the anti-Catcher antibody spiked with sodium azide, the membranes were detected using ECL to see if there was still functional HRP from the detection of the first antibody. The sodium azide inactivates the remaining HRP. HPR - horse radish peroxidase.*

**Results:**
After re-incubation with a secondary mouse antibody, bands at the level of ~55 kDa are now detectable as well as bands at 100 kDa (Fig. 4.22-2.25).

![Figure 4.22: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pAzFRS, and pCDFDUET-1](https://static.igem.wiki/teams/4229/wiki/pictures/re-ncaa-422.png)

***Figure 4.22: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pAzFRS, and pCDFDUET-1.** The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used as ladder. For detection, an α-Catcher antibody (1:1000) was used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, pAzF – p-azido-l-phenylalanine.*

![Figure 4.23: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pAzFRS, and sTAF sXTB. ](https://static.igem.wiki/teams/4229/wiki/pictures/re-ncaa-423.png)

***Figure 4.23: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pAzFRS, and sTAF sXTB.**  The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used as ladder. For detection, an α-Catcher antibody (1:1000) was used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, pAzF – p-azido-l-phenylalanine.*

![Figure 4.23: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pAzFRS, and sTAF sXTB. ](https://static.igem.wiki/teams/4229/wiki/pictures/re-ncaa-424.png)

***Figure 4.24: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pBpF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pBpFRS, and sTAF sXTB.** The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used as ladder. For detection, an α-Catcher antibody was used (1:1000) in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, pBpF – p-benzoyl-l-phenylalanine.*

![Figure 4.25: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pBpF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pBpFRS, and sTAF sXTB. ](https://static.igem.wiki/teams/4229/wiki/pictures/re-ncaa-425.png)

***Figure 4.25: Western blot of an SDS-PAGE with whole cell lysis confirming the incorporation of pBpF at position 35 in the BMC-T1 protein with the transformation of BMC-T1-T35TAG, pEVOL-pBpFRS, and sTAF sXTB.** The cells grew until OD600 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used as ladder. For detection, an α-Catcher antibody (1:1000) was used in combination with an anti-mouse-HRP secondary antibody (1:10000). IPTG - Isopropyl β-D-1-thiogalactopyranoside, pBpF – p-benzoyl-l-phenylalanine.*

### Conclusion

The bands at 55 kDa fit well with the expected size of the band, as XiaI (Indigo enzyme) has a size of 42.5 kDa and the spyCatcher has a size of 12.3 kDa, which should be detectable at 55 kDa. If the empty backbone of the indigo enzymes is transformed, no band is visible. However, if you transform the plasmid with the enzymes but do not express it, you see a weak band at 55 kDa, which suggests leakiness. If the enzymes are expressed, strong bands are seen at 55 kDa, but also below, which indicates degradation. When the full wiffleball is expressed together with the indigo enzymes, a band at ~100 kDa can be seen, which was also detected by the 6xHisTag antibody. This should be the BMCs that captured the indigo enzymes. However, you can see that there is still a strong band at 55 kDa, which suggests that not every enzyme is captured. Since we saw the same for the 6xHisTag antibody, there are both BMCs and indigo enzymes in the cell that are not catching each other.
For the BMC mutants that were not transformed with sTAF sXTB (Fig. 4.22 + 4.24), light bands are seen for the non-induced samples, but not for the induced samples. Here it looks like a little of the other sample has leaked into the pocket before running the SDS-PAGE. We have also seen previously that it can bind antibodies non-specifically, and we are mainly interested in the samples where both the indigo enzymes and the mutant BMC were expressed.
In the samples in which both the mutant BMC and the indigo enzymes were expressed (Fig. 4.23 + 4.25), a band at the level of 100 kDa is also seen. However, the same phenomenon is seen as with the unmutated BMCs and indigo enzymes: not all enzymes seem to be captured. Accordingly, the stoichiometry of the proteins would have to be refined, but we can prove that our enzymes are captured despite the mutation.

## Pathways

---

### Indigo/Indirubin pathway

**Key findings:**

* With the help of three different techniques, we were able to prove that it is indeed indigo and indirubin that we produce.
* We have successfully incorporated our enzymes into the full wiffleball.
* We have successfully created constructs that include our pathway, mutated wiffleball (with the T1 protein bearing the amber stop codon) and the incorporation of ncAAs using the pAzF synthetases.
* We have successfully produced indigo on a large scale by using a bioreactor.

## Design of the expression plasmid

The first step to work on the production of indigo was to design the plasmid that we wanted to use. We have decided to use the genes that were used by Yin et al. [[5.1]](#re_ref5_1) for the production of indigo and indirubin in *E. coli*.

![Figure 5.1: Schematic representation of the indigo/indirubin pathway.](https://static.igem.wiki/teams/4229/wiki/pictures/design/ds-indigo-pathway-schematic.jpg)

***Figure 5.1: Schematic representation of the indigo/indirubin pathway.**L-Tryptophan is imported by the membrane protein TnaB (low affinity tryptophan permease). L-tryptophan is cleaved into indole, NH4+ and pyruvate by the Tryptophanase TnaA. The reaction continues by the hydroxylation of indole through XiaI. To enhance the effectivity of this enzyme, the NAD(P)H-flavin reductase provides XiaI with FADH2 by adding Hydrogen to FAD. Finally, indole is transformed to either 3-Hydroxyindole or 2-Hydroxyindole. These two substances spontaneously react to 3-Oxindole and 2-Oxindole through the secession of hydrogen from the OH-group. Through spontaneous dimerization indigo and indirubin are formed. Graphic adapted from [[1.2]](#re_ref1_2).*

These genes are TnaA, Fre, XiaI and TnaB (protein sequence information available on UniProt [2][3][4][5]), which were codon optimized for expression in *E. coli* using the IDT-codon-optimization tool.
We decided to use pCDFDUET-1 as our backbone as it is designed for the coexpression of two target genes. The vector encodes two multiple cloning sites (MCS) each of which is preceded by a T7 promoter, lac operator, and ribosome binding site (rbs). Thereby only one plasmid is needed for the full pathway, which can be later be combined with the compartment systems (wiffleballs, EncA, SPD5) and the ncAA incorporation system.
For the design of the expression construct, we paired two genes together as follows: The first gene block consisted of TnaA and Fre (referred to as TAF) and was included into the MCS1. The second block consists of XiaI and TnaB (referred to as XTB) and was included into the MCS2. A ribosome binding site (RBS) was added between the two genes of each MCS.
To target the pathway to the compartments we added the SpyCatcher sequence to XiaI and the SnoopCatcher sequence to TnaA as these enzymes are the ones supposed to be incorporated into our compartment systems (wiffleball or SPD5). Two catcher constructs were designed, having attached the Catcher sequences either attached to the C-terminus or N-terminus of XiaI and TnaA . We added GS-Linkers between enzyme and catcher sequences. (Figure 5.2)

![Figure 5.2: Schematic representation of the designed pathway constructs.](https://static.igem.wiki/teams/4229/wiki/pictures/results/indigo/2-plasmid.png)

*Figure 5.2: Schematic representation of the designed pathway constructs.Via restriction enzymes, gene fragments were cloned in the pCDFDuet-1 backbone. A ribosomal binding site was inserted between the two target genes of one MCS.*

The following constructs were generated:

| Plasmid name | Description |
| --- | --- |
| pCDFDuet-TAF-XTB | TnaA and Fre inserted in MCS1   XiaI and TnaB inserted in MCS2 |
| pCDFDuet-sTAF-sXTB | TnaA with N-terminal SpyCatcher and Fre inserted in MCS1   XiaI with N-terminal SnoopCatcher and TnaB inserted in MCS2 |
| pCDFDuet-TAFs-XTBs | TnaA with C-terminal SpyCatcher and Fre inserted in MCS1   XiaI with C-terminal SnoopCatcher and TnaB inserted in MCS2 |
| pCDFDuet-sTAF-XTBs | TnaA with N-terminal SpyCatcher and Fre inserted in MCS1   XiaI with C-terminal SnoopCatcher and TnaB inserted in MCS2 |
| pCDFDuet-TAFs-sXTB | TnaA with C-terminal SpyCatcher and Fre inserted in MCS1   XiaI with N-terminal SnoopCatcher and TnaB inserted in MCS2 |

Due to time constrains, we could not compare all the different constructs to each other to see which one works best. Because of that, we decided to use pCDF-DUET 1-snoopTnaA-Fre-spyXiaI-TnaB (Figure 1 B) for all of our assays, as this construct was the first one to be finished. We have used this construct for each assay.

### Experimental design

Throughout our project, we have done several assays testing the production of indigo and indirubin under different conditions. To learn more about our procedures click here, to learn about the protocols that we have used or click here to get to our LabBook, where you can retrace our experiments.
All of our assays were performed in triplicates. This allowed us to create a mean value for each condition that we have tested. Furthermore, this significantly lowers the risk of human errors that could falsify our data and we can recognize outliers with much more precision. By making triplicates of all conditions, we can ascribe our final data with more weight.
Additionally, we have designed our assays as self-contained experiments. We planned our experiments to test an assumption, and the evidence to evaluate this assumption always emerged from one single assay. That way we could make sure, that effectivity differences stem from the tested conditions, and not because of a slightly different experimental environment.
By combining the use of triplicates together with a closed, self-contained design for each experiment testing an assumption, as underlying concepts for every single assay, we produced data that is as meaningful as possible.

### Proof of the production of Indigo and Indirubin

**Aim:** Verify that our bacteria can produce indigo and indirubin by recombinant overexpression of XiaI, TnaA, TnaB and Fre in *E. coli* BL21(DE3).

**Experimental setup:** According to our [Indigo/ Indirubin production protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D), we collected samples of our E. coli BL21 transformed with the pCDFDuet-1-sTAF-sXTB plasmid and tested for indigo and indirubin production via photometric measurements, NMR and mass spectrometry. A detailed protocol for each step of sample preparation can be found [here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).

**Results:** We successfully produced indigo and indirubin with our designed constructs, pCDFDuet-1 sTAF-sXTB, what we could clearly see by a colour change of liquid cultures 24 hours after induction (see [Fig. 5.3](#re_fig5_1)). Following sample preparation, we measured the absorbance/fluorescence of both compounds at their respective absorption peaks (see [fig. 5.6](#re_fig5_6)) for all our production assays.

![Indigo and Indirubin from own bioproduction using E. coli BL21 pCDFDuet-1 TAsF-sXTB, colouring the medium bluish (through Indigo) or reddish (through Indirubin).](https://static.igem.wiki/teams/4229/wiki/pictures/results/indigo/re-indigo-indirubin-ownproduction.jpg)

***Figure 5.3: Liquid cultures of E. coli BL21(DE3) transformed with pCDFDuet-sTAF-sXTB, showing the production of indigo and indirubin.**  A: Left erlenmeyer flasks with 200 mL culture of indigo producing bacteria, right erlenmeyer flasks with 200 mL culture of indirubin producing bacteria; B: Pellet of 1.5 mL bacterial culture after indigo production; C: Pellet of 1.5 mL bacterial culture after indirubin production. To shift the reaction from indigo to indirubin production cultures were supplemented with L-Cysteine.*

Measuring proton NMR spectra of indirubin from our bioproduction shows small peaks at a shift of 7 – 7.6 ppm (see [Fig. 5.4](#re_fig5_4)), which roughly fits the expected spectrum of indirubin from the literature [[5.1]](#re_ref5_1). However, the peaks are too weak to reveal any further information. This might be due to too high contamination with other cell substances, as we did not have the equipment to purify further our samples. The most prominent peak at ~4 ppm is most likely from water, and the peak at ~2.5 ppm from DMSO. To prevent the massive water contamination, we freeze-dried the indigo sample indigo before measuring. It might also be that the shift to indirubin production, by adding L-cysteine, was not efficient enough to produce detectable amounts. However, NMR clearly shows successful production of indirubin.
![NMR proton-spectrum of Indirubin from own bioproduction using E. coli BL21 pCDFDuet-1 sTAF-sXTB. ](https://static.igem.wiki/teams/4229/wiki/4-nmr.png)

**Figure 5.4: NMR proton-spectrum of indirubin from bioproduction using pCDFDuet-sTAF-sXTB E. coli BL21(DE3).** Indirubin-producing bacteria were lysed, the non-soluble pellet was, after washing with KPI buffer, resuspended in deuterated DMSO for measurement. B shows a zoomed-in section of A.

Additionally, we have performed HLPC separation of indirubin from our samples and detected the molecular mass using mass spectrometry [(Fig. 5.5)](#re_fig5_5).
![HPLC-followed mass spectrometry of Indirubin from own bioproduction using E. coli BL21 pCDFDuet-1 sTAF-sXTB.](https://static.igem.wiki/teams/4229/wiki/pictures/results/indigo/re-indirubin-hplc-followed-massspec.jpg)

**Fig. 5.4: HPLC-followed mass spectrometry of Indirubin from own bioproduction using *E. coli* BL21 pCDFDuet-1 sTAF-sXTB.**Indirubin-producing bacterial culture was spun down; the pellet was resuspended in KPI buffer, sonicated, spun down again and resuspended in deuterated DMSO for measurement. A: HPLC diagram, B: mass spectrometry diagram.

### Calibration Curves using synthetic Indigo and Indirubin

**Aim:** To calculate the concentration of indigo and indirubin in our bioproduction photometrically, we needed standard curves to compare our measurements.

**Experimental setup:** Using synthetic indigo and indirubin (commercially available from Carl Roth), we set up different calibration curves according to the [calibration-curves protocol](protocols.html#pr_indigo). Absorbance of indigo and indirubin was measured at 540 nm. Additionally, fluorescence of indigo was measured at 612 nm excitation and 670 nm emission, using a plate reader. These experiments were performed in triplicates. For Indigo measurements, the experiments were repeated additionally with lower concentrations to obtain a better resolution for a possibly low indigo production.

**Results:**

![Calibration curves with synthetic Indigo and Indirubin.](https://static.igem.wiki/teams/4229/wiki/pictures/results/indigo/re-indigo-indirubin-calibrationcurves-largescreen.jpg)
![Calibration curves with synthetic Indigo and Indirubin.](https://static.igem.wiki/teams/4229/wiki/pictures/results/indigo/re-indigo-indirubin-calibrationcurves-smallscreen.jpg)

**Fig. 5.6: Calibration curves with synthetic Indigo and Indirubin.**  Absorbance at 540 nm was measured for indigo (A) and indirubin (C), as well as fluorescence of indigo with excitation at 612 nm and emission at 670 nm (B).

The calibration curves (see [Fig. 5.6](#re_fig5_6)) were performed successfully, showing a linear increase of the relation of absorbance/fluorescence to the concentration for all tested conditions. using excel linear regression calculator allowed us to calculate the concentration of indigo/indirubin, by using the following formulas (with x = concentration (µM), y = absorbance (a.u.):

* Indirubin absorbance 540nm: 0.0059 \* x = y
* Indigo absorbance 540nm: 0.0048 \* x = y
* Indigo fluorescence: 758.95 \* x = y

With this, we calculated the produced indigo and indirubin concentration for our samples by replacing the measured absorbance/fluorescence for each of the following production assays.
However, as the absorbance spectra of indigo and indirubin interfere with each other, only the indigo concentration could be calculated directly from the fluorescence with the corresponding formula. With the estimated indigo concentration, we could calculate the fitting absorbance that indigo must have at 540 nm, subtracting the calculated absorbance from the absorbance measurement at 540 nm and getting only the absorbance from indirubin. We could then enter the corresponding formula for this value to calculate the totalindirubin concentration in the samples.
We also considered that the IPTG concentration might influence our production assays and eventually falsify the measured of absorbance/fluorescence. Therefore, we tested different concentrations of IPTG (up to 2 mM) showing no effect on the photometric output.
One interesting observation we did was that for indigo concentrations of >30 µM, the fluorescence was not going higher in a linear matter but stagnated first and then went down again (see in the [labbook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27labbook%27%29%20%7D%7D) in our raw data). At concentration of more than 30 µM, the solution of indigo was so dark assuming that too much light is absorbed and thereby less fluorescence can be measured. With this, we knew that samples need to be diluted before fluorescent measurement.

### Optimisation of IPTG and L-Trp concentration for the pathway

**Aim:** Test different IPTG and L-Tryptophan (L-Trp) concentrations to find the most suitable one for the highest production of indigo/indirubin. IPTG is needed to induce the pathway promoter (T7-promoter), and L-Tryptophan is the substrate of the pathway.

**General experimental setup:** We used *E. coli* BL21 with pCDFDuet-1 sTAF-sXTB (exclusively containing the pathway) for the following experiments. All conditions were tested in triplicates, following the [Indigo/Indirubin-production protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) and the [Indigo/Indirubin-quantification protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).

**Experimental setup IPTG optimisation:** We first induced samples with varying IPTG concentrations (from 0 to 0.15 mM final concentration). Following induction at OD=0.8 5 mM L-Trp was added temperature was set to 25°C for further culture. The tested IPTG concentration we used based on the experimental data published by Yin et al. [[5.1]](#re_ref5_3). Moreover, O2 supply was also highlighted by Yin et al. to be an essential factor for yield increase. By using 500 mL Erlenmeyer flasks for the 200 mL bacterial cultures, we could enhance the aeration of the cultures and guarantee sufficient O2 supply.

**Results IPTG optimisation:** Several experiments were done to test for optimal IPTG concentration (see labbook). In this preselected area, we found out that there is a tendency for 0.1 mM of IPTG in medium to work best to produce high yields. Nevertheless, 0.2 mM IPTG concentration seems to be nearly similarly effective. A more significant difference occurs 24h after induction (Figure 5.7). Production of indigo/indirubin was detected following induction with IPTG. Already 0.02 mM ITPG significantly leads to the production of about 2.5 µM indigo, 30 hours after induction. There were no major increase in indigo detected with longer expression time (3 µM after 45 hours).
A significant productionof indirubin of about 20 µM was determined 30 hours after induction. The highest concentration of indirubin (~50 µM) was obtained after 36 hours with no further increase with culture time. There was no major difference for the tested IPTG concentrations.
An interesting observation was that we already produced higher Indirubin amounts than Indigo while not switching the reaction by adding cysteine to the culture (see the comparison between A and B under the same conditions in fig.).
In comparison to our initial trial (see lab book fig.) in this experiment (see fig.), we could also see the positive effect of better O2 supply, like Yin et. al described [1]. In our initial experiment we only used 250 mL erlenmeyer flasks for culturing 200 mL bacteria, while in this assay we used 500 mL erlenmeyer flasks. Thereby the bacterial cultures had more contact surface with the air while shaking and should be able to take up more O2.
(see [Fig. 5.7](#re_fig5_7)).
![Optimisation of IPTG-concentration for the Indigo/Indirubin pathway using E. coli BL21 pCDFDuet-1 sTAF-sXTB (pathway-enzymes with catcher).](https://static.igem.wiki/teams/4229/wiki/pictures/results/indigo/re-productionassay-iptg-optimization.jpg)

**Fig. 5.7: Optimisation of IPTG-concentration for the Indigo/Indirubin pathway using *E. coli* BL21 pCDFDuet-1 sTAF-sXTB (pathway-enzymes with catcher).** A: Indigo-concentration measured *via* fluorescence (excitation 612 nm, emission 670 nm), B: Indirubin-concentration measured *via* absorbance at 540 nm. Production with 200 mL cultures in 500 mL Erlenmeyer-flasks, at 25 °C after IPTG-induction at OD = 0.8, with L-Tryptophan-concentration of 5 mM. Photometric readout using a plate reader.

From this assay, we concluded to use 0.02 mM IPTG to induce the expression of pathway genes. This concentration is enough to get a reasonable amount of product and not too high to stress the cells. Moreover, higher IPTG concentration seem to have to further beneficial effect on the production of indigo/indirubin.

**Experimental procedure L-Trp optimisation:** For the L-Trp optimisation, we tested concentration between 0 and 10 mM L-Trp. The IPTG concentration of 0.02 mM was fixed for this experiment. The conditions (25 °C after L-Trp induction at OD = 0.8, 500 mL Erlenmeyer flasks for the 200 mL bacterial cultures) stayed the same as in the experiment before.

**Results L-Trp:** A concentration of L-Trp of 2.5 mM worked the best to produce high yields (~70 µM) of indirubin (see fig. 5.8 B). Higher concentrations (>7.5 mM) of L-Trp have no further positive effect; production of indirubin is comparable to that obtained without L-Trp supplementation (Fig…B, 0 vs 10 mM). Indigo production also increased following L-Trp supplementation. The highest difference was obtained compared to non-supplemented condition was obtained 18 to 30 hours after induction (Fig. 5.8 A).
Interestingly, cells already produced Indigo and Indirubin without L-Trp addition, probably due to cell’s own L-Trp consumption. However, with the addition of additional L-Trp, the cells produced more indigo/indirubin.
As observed in the IPTG optimisation assay, more Indirubin was produced than Indigo, ~4.5 fold (see the comparison between A and B under the same conditions in fig. 5.8). The highest production yield could also be improved compared to the IPTG optimisation assay, most likely because now most optimal tested IPTG and L-Trp concentrations worked together in at least one condition (see the best condition with 2.5 mM L.Trp and 0.02 mM IPTG concentration in fig. 5.8).
![Optimisation of L-Tryptophan-concentration for the Indigo/Indirubin pathway using E.coli BL21 pCDFDuet-1 sTAF-sXTB (pathway-enzymes with catcher).](https://static.igem.wiki/teams/4229/wiki/pictures/results/indigo/re-productionassay-ltrp-optimization.jpg)

***Fig. 5.8: Optimisation of L-Tryptophan-concentration for the Indigo/Indirubin pathway using E.coli BL21 pCDFDuet-1 sTAF-sXTB (pathway-enzymes with catcher).***A: Indigo-concentration measured via fluorescence (extinction 612 nm, emission 670 nm), B: Indirubin-concentration measured via absorbance at 540 nm. Production with 200 mL cultures in 500 mL Erlenmeyer-flasks, at 25 °C after IPTG-induction at OD = 0.8, with IPTG-concentration of 0.02 mM. Photometric readout using a plate reader.

### Conclusion & Outlook

We could verify and measure the production of Indigo and Indirubin, and calculate with the help of calibration curves, how much Indigo and Indirubin was produced. We could further test, which IPTG and L-Trp concentrations work best for our construct to reach optimal levels of Indigo of ~16 µM, and ~73 µM of Indirubin.
As we were more interested in the effects of compartment systems, we focused more on the combination of both together instead of further optimizing the pathway. Here still a lot could be done. E.g. a comparison of different Spy-Snoop-catcher positions could be tested and importantly also with a no-Catcher construct, to see the influence of the catchers on the pathway itself. Furthermore, as we saw hints in our first experiments and from the literature of the importance of O2 supply for higher yield, different O2 concentrations could be tested. And many more screws could be turned to increase the yield. One last to mention, which would be very interesting for shifting the reaction to Indirubin, is, how much L-Cysteine is needed or optimal for this.

## Trehalose pathway

**Key findings:**

* We successfully designed and cloned plasmids containing the two key enzymes of the Trehalose pathway
* We have shown *in vivo* trehalose production in *E.coli*

### Plasmid Design

**Aim:** Our goal was to clone the genes *otsA* and *otsB* from the *E. coli* genome and insert them into pCDF-Duet-1. Later, we switched to the pET-Duet-1 backbone.
![Plasmids we wanted to make.](https://static.igem.wiki/teams/4229/wiki/trehalose-final-1.png)

**Figure 6.1: Those are the plasmids we wanted to make.** pCDF-Duet-1 was chosen as we already used it for the Indigo pathway. Both plasmids have two MCS (multiple cloning sites) and are regulated by a T7 promotor.

**Experimental setup:** We first started by amplifying *otsA* and *otsB* out of the genome using olG22\_001 (ATTACCATGGatgAGTCGTTTAGTCGTAGTATCTAACCGG) and olG22\_002 (ATTAGCGGCCGCctaCGCAAGCTTTGGAA) for *otsA* and olG22\_003 (ATTACATATGatgacagaaccgttaaccgaaacc) and olG22\_004 (ATTACTCGAGttagatactacgactaaacgact) for *otsB*.
![PCR for amplifying otsA and otsB out of the genome of E. coli.](https://static.igem.wiki/teams/4229/wiki/pictures/results/trehalose/re-gelotsabpcr.jpg)

**Figure 6.2:** PCR for amplifying *otsA* and *otsB* out of the genome of *E. coli* using primer olG22\_003 and olG22\_004 for *otsB* and olG22\_001 and olG22\_002 for *otsA* and different temperatures.

*otsA* and *otsB* were then subcloned into the subcloning vector pJET1.2 with the provided kit.
![Vector maps of the subcloned otsA and otsB. ](https://static.igem.wiki/teams/4229/wiki/trehalose-final-2.png)

**Figure 6.3: Vector maps of the subcloned *otsA* and *otsB*.**

*otsB* was successfully subcloned and sequenced. *otsA* initially caused us difficulties but was also successfully subcloned. This was shown by colony PCR (Fig. 6.4) and subsequent sequencing.
![Colony PCR made from subcloning otsA into pJet1.2.](https://static.igem.wiki/teams/4229/wiki/pictures/results/trehalose/re-gelotsacolony.jpg)

**Figure 6.4: Colony PCR made from subcloning *otsA* into pJet1.2. with a length of 1566 between the sequencing primers.**

After the successful subcloning we tried to clone *otsB* into pCDF-Duet-1. This did not work, so we decided to use pET-Duet-1. We did this by digesting pJet1.2+ots and pET-Duet-1 with the restriction. enzymes XhoI and NdeI.
![An enzyme digest for pJet1.2 + otsB and pCDF-Duet-1 to prepare it to be ligated into the MCS2.](https://static.igem.wiki/teams/4229/wiki/pictures/results/trehalose/re-gelotsbdigest-2.jpg)

**Figure 6.5: An enzyme digest for pJet1.2 + *otsB* and pCDF-Duet-1 to prepare it to be ligated into the MCS2.**Both constructs were digested with NdeI and XhoI.

Which was then ligated by a T4 ligase. That was also successful as seen in the following colony PCR.
![Colony PCR made from the ligation made on 09.07.2022.](https://static.igem.wiki/teams/4229/wiki/pictures/results/trehalose/re-gelpetotsb.jpg)

**Figure 6.6: Colony PCR made from the ligation made on 09.07.2022.** Here are the colonies from pET-Duet-1 + *otsB*. Colonies with a band running at 800bp were positive.

After also sequencing it, we would start to clone *otsA* into pET-Duet. That was also made by restriction enzyme digest. This time the restriction enzymes NcoI and NotI were used.
![Two gels with pJet1.2 + otsA and pET-Duet-1+  otsB were digested with NcoI and NotI to ligate otsA into the MCS2.](https://static.igem.wiki/teams/4229/wiki/pictures/results/trehalose/re-gelotsadigest1.jpg)

**Figure 6.7: Two gels with pJet1.2 + *otsA* and pET-Duet-1 + *otsB* were digested with NcoI and NotI to ligate otsA into the MCS2.**

We ligated them by using a T4 ligase. And after a heat shock traffold into chemically competent TOP10 E. coli we also successfully cloned *otsA* into pET-Duet-1 +otsB. The sequencing showed also a positive result.
![Colony PCR with pET-Duet-1 +otsA +otsB.](https://static.igem.wiki/teams/4229/wiki/pictures/results/trehalose/re-gelpetotsabcolony-2.jpg)

**Figure 6.8: Colony PCR with pET-Duet-1 + *otsA* + *otsB*.** Colonies with a band running at 1500bp were positive.

**Results:**
We have successfully cloned the plasmid pET-Duet-otsAB. For the later detection of trehalose, two glycerol stocks were created. First, *E. coli* BL21 transformed with pET-Duet. And secondly, *E. coli* BL21 transformed with pET-Duet-otsAB.

### Quantity / Amount of Trehalose

**Aim:** To quantify the production of trehalose, a calibration curve was first performed using different trehalose concentrations.

**Experimental setup:** The calibration curve was performed with the Trehalose Assay Kit from Megazyme. The protocol for the microplate assay procedure was followed. Lysed *E. coli* BL21 served as sample. Different amounts of trehalose were added (0 µg, 1 µg, 2 µg, 4 µg, 6 µg, 8 µg and 10 µg).

**Results:**
The trehalose present is hydrolysed by the enzyme trehalase to 2 D-glucose. In the presence of ATP, the added hexokinase phosphorylates the D-glucose to glucose-6-phosphate. The enzyme glucose-6-phosphate dehydrogenase oxidises this to gluconate-6-phosphate in the presence of NADP+. This produces NADPH + H+. The presence of NADPH can be seen in the increase in absorbance at 340 nm and thus provides a detection for trehalose. The calibration curve was plotted using Excel (Microsoft Office 365 Pro Plus). The indicated error corresponds to the standard error of the mean (SEM). The calibration curve is shown in Figure 6.9.
![Calibration curve of trehalose.](https://static.igem.wiki/teams/4229/wiki/pictures/results/trehalose/re-trehalose-calibrationcurve.png)

**Figure 6.9: Calibration curve for trehalose.** Different amounts of trehalose were measured (0 µg, 1 µg, 2 µg, 4 µg, 6 µg, 8 µg and 10 µg). The error bars correspond to the standard error of the mean. Plotted using Excel 365.

### Best growth conditions

**Aim:** We want to find out the best conditions for the growth of *E. coli* BL21.

**Experimental setup:** In order to find out the best growth conditions for *E. coli* BL21, we performed a growth curve in LB medium with different concentrations of glucose. Since trehalose is endogenously synthesised under osmotic stress, we decided to additionally test different salt concentrations. For this purpose, a 96-well plate was used and measured in the plate reader for over 17 hours.

**Results:**
 Figure 6.10 shows the growth curve. The absorbance was plotted against time. The sample without glucose reached a plateau earliest after 180 minutes and continued to grow slowly thereafter. The samples with glucose reached their maximum OD after 330 to 360 minutes. The sample with 4% glucose reached the highest OD with 0.793. However, since a sample with 3% reached a lower OD with 0.762 than those with even less glucose, i.e., 1 and 2%, we decided against the sample with 4% glucose. Since the sample with 2% glucose showed slower growth with comparable maximum (0.79 for 1% glucose, 0.78 for 2% glucose), we decided to use 2% glucose for the production assays.
The samples with added salt showed less growth. Furthermore, they showed a partial fluctuation in their course. As expected, the samples with a higher salt concentration showed a lower OD.
![Growth curve of E. coli BL21.](https://static.igem.wiki/teams/4229/wiki/pictures/results/trehalose/re-trehalose-growthcurve.jpeg)

**Figure 6.10: Growth curve of *E. coli* BL21.** Growth conditions in LB medium with different glucose concentrations (0-4%) were investigated. Different concentrations of NaCl (200-600 mM) were added to three samples with 2% glucose. The error corresponds to the standard deviation.

### Improve of Trehalose

**Aim:** We want to detect the production of trehalose in the transformed *E. coli* BL21.

**Experimental setup:** The growth and trehalose production of the transformed *E. coli* BL21 were investigated in different media. We tested bacteria with the empty backbone pET-Duet and our plasmid pET-Duet-otsAB. We decided to use LB Medium with 2% Glocose as described. The bacteria were incubated at 37 degree and induced with 0.5 mM IPTG from an OD of 0.6. After that, a sample was taken every two hours. The last sample was taken after 12 hours. The samples were centrifuged, washed with 1 x PBS and resuspended in ddH2O. Lysis was carried out by sonication. The production assay was performed with the Trehalose Assay Kit from Megazyme. The protocol for the microplate assay procedure was followed.

**Results:**
Using the kit, trehalose production can be detected as described. The result is shown in [Figure 6.11](#re_fig6_11). The absorbance was plotted against time. The induced sample containing the plasmid pET-Duet-otsAB shows a maximum of 0.654 after 4 hours of incubation. After reaching the maximum, the absorbance subsequently decreases until after 12 hours it is only slightly above the other samples. The uninduced plasmid, as well as only the backbone (induced and uninduced) shows a significantly lower absorbance of about 0.32. This allowed us to show the production of trehalose. And thus, also confirm the functionality of our plasmid. We were able to detect a mass of 2 to 4 µg Trehalose.
![Trehalose detection after productionassay.](https://static.igem.wiki/teams/4229/wiki/pictures/results/trehalose/re-trehalose-productioncurve.png)

**Figure 6.11: Trehalose detection after productionassay.**  *E. coli* BL21 with the plasmids pET-Duet and pET-Duet-otsAB induced and uninduced were tested for the production of trehalose within a 12-hour incubation. Only the IPTG-induced bacteria with the plasmid pET-Duet-otsAB reach a maximum of 0.654 after 4 hours. The indicated error corresponds to the SEM.

### Outlook

Our next step is to introduce catchers into our plasmid. This involves adding a spy catcher to otsA and a snoop catcher to otsB. After that, it is possible for the trehalose pathway to take place in our Wiffleball. After fine-tuning, we can hopefully achieve a higher efficiency for our production.

## Genome Reduced Bacteria

---

**Key findings:**

* The growth of genome reduced strains is heavily limited at low temperatures, e.g. 18°C
* We can reliably incorporate non-canonical amino acids into sfGFP in different E. coli strains

### **Wiffleball expression in genome reduced strains**

**Aim:** Testing the wiffleball system in different genome-reduced *E. coli* strains to check their specific behavior, their protein expression and metabolic burden.

**Experimental design:** We tested the full and minimal wiffleball, besides the already tested BL21(DE3), MG1655 and C321∆exp, in ME5119, a derivate of MG1655, which has 15.8% of its genome deleted, and in MDS69 LowMut T7, a commercial genome reduced strain von Scarab Genomics with 20.32% deleted genome, with the same experimental conditions as in the previous induction experiment. The cells were treated with different inducer concentration reaching from 100µM up to 1mM IPTG and validated the expression with fluorescence microscopy for the foci formation, western blot for the protein expression level and growth assays for the metabolic burden. For the growth assay, the bacteria were induced at OD600= 0.1 in a 96-well plate with all different inducer concentrations and incubated at 30°C, shaken in a plate reader for 20h. These conditions were also changed due to the lack of a cooling function to 18°C of the device, limited use of the plate reader and a restricted maximum growth to OD600= 1.4 due to the 200µl well volume. For comparison between the strains, they are shown in the same plots and were measured on the same 96-well plate.

**Results:** For our goal of using genome reduced bacteria in bioproduction, we tested ME5119 and MDS69 LowMutT7. Both strains showed at 18°C a slower growth compared to MG1655 and BL21(DE3) Furthermore, the fluorescent expression of mVenus2 of ME5119 and LowMutT7 was also less than in all other strains. When the BMCs were expressed, we could not observe any foci in both strains and no bands of T1 could be detected on the western blots. Cell division and induced protein expression at 18°C seem to be a large burden for these strains. The experiments were repeated twice for ME5119.

The growth assay of ME5119 showed an overall trend: the higher the inducer concentration, the less the growth (Figure 8.1). In the growth comparison in the plate reader at 30°C, ME5119 grew remarkably slower than MG1655 and BL21. (Figure 8.2)

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-27-1-me-full.png)
![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-27-2-me-min.png)

**Figure 8.1: ME5119 growth in plate reader at 30°C with different inducer conditions. In legend: Wiffleball (IPTG (µM)/Doxycylcine (ng/ml))**

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-28-growth-me-bl-mg.png)

**Figure 8.2: Growth comparison in plate reader at 30°C between BL21(DE3), MG1655 and ME5119 with induction of 100µM IPTG 25ng/ml doxycycline**

LowMut T7 was hardly influenced by IPTG concentrations lower than 400µM (Figure 1.29). For the minimal wiffleball induction, IPTG concentrations higher than 400µM had only slight impact on the growth (Figure 8.3).

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-29-1-lowmut-full.png)
![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-29-2-lowmut-min.png)

**Figure 8.3: LowMut T7 growth in plate reader at 30°C with different inducer conditions,**

By observing the growth curve of both genome reduced strains, we noticed a further difference between the two: ME5119 never reaches the OD600 of 1, while LowMutT7 goes a bit further. At this point, we were curious to compare this strain to our well working BL21(DE3), so we repeated the same experiment under the same conditions, inducing both BL21(DE3) and LowMutT7 on the same 96-well plate. Surprisingly, as shown in Figure 1.30, no big difference in the growth course of the compared strains could be detected.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-30-growth-bl-lowmut.png)

**Figure 8.4: Growth comparison in plate reader at 30°C between BL21(DE3) and LowMut T7 with induction of 100µM IPTG and 25ng/ml doxycycline**

For more data of the growth assays check our supplementary data in the labbook.

### **Testing longer induction times**

**Aim:**In previous we saw that the growth of the genome reduced strain ME5119 was strongly reduced compared to MG1655 or BL21(DE3). This was accompanied by lower expression of Wiffleballs, we therefore test longer induction time trying to enhance the Wiffleball expression.

**Experimental design:**The already used induction conditions for the wiffleball induction at OD600= 0.6-0.7 with incubation at 18°C, 200rpm, stayed the same. We took samples from the induced cultures from different time points, after 24h and 48h from ME5119. We examined the foci formation and protein expression by fluorescent microscopy and western blotting as in the wiffleball experiments before.

**Results:**

ME5119 showed low fluorescence under the microscope after 24h of incubation. No foci were observable. Even after 48 hours, the results stayed the same. We could not detect any T1 protein in the samples by western blot (data can be found in the supplementary).

**Conclusion:**

Longer incubation times had no effect on the overall protein expression on ME5119.

### **Adjusting incubation temperature**

**Aim:** The expression and formation of the wiffleball under high temperatures results in larger amount of unfunctional inclusion bodies. Therefore, we always expressed the wiffleball at 18°C. Through an interview with Erik, from the Kerfeld Lab, we received the valuable information of incubating our cells after induction for 3h at 37°C, followed by the typical incubation temperature at 18°C for 16-21h. This should boost our protein expression, together with properly formed compartments.

**Experimental setup:** Following this new advice, we induced our BL21(DE3) and the genome reduced LowMut T7 at OD600 = 0.6-0.7 with 100µM IPTG for the full and 400µM IPTG for the minimal wiffleball with additional 50ng/ml Doxycycline for mVenus2. The first incubation step for 3h incubation at 37°C, followed by 21h at 18°C, all shaken for 200rpm.

Samples were harvested after 24h of whole incubation time and then put back in the incubator.

The observation of the samples took place by the already used fluorescence microscopy method and western blot.

Unfortunately, the western blot will be detected after the wiki freeze due to the limited time.

**Results:**

Change the incubation conditions resulted in a remarkable change visible under the microscope. Almost every BL21(DE3) cell with the full wiffleball construct contained foci. Quantification for this will follow the wiki freeze, but by eye the estimation is a nearly two-fold increase of the number of foci. Some cells even showed a second polarised focus, pushing right toward the pole against the first focus. After 48h of induction, the number of secondary foci increased (Figure 8.5 A, red arrows). However, these results must be treated with care since we first need to exclude the possibility of inclusion bodies by western blot.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/1-31-bl21-lowmut-24h-48-comparison.png)

**Figure 8.5 :** adjusted incubation temperature at 37°C for 3h followed by 21h at 18°C; full wiffleball induced with 100µM IPTG, minimal wiffleball induced with 400µM lPTG, mVenus2 induced with 50ng/ml; A BL21(DE3); B LowMut T7

In the previous experiment, LowMut T7 had big troubles growing at 18°C and expressing our non-native proteins. The new expression conditions changed their behavior drastically. The bacterial cultures were much denser than the previous induced LowMut T7 cultures. Also, under the microscope the change was drastic. With the full wiffleball, a lot of bacteria were showing the foci structures toward one cell pole. After 48h, the number of foci increased and were sometimes additionally localised in the middle of the cell.

The results seen in the minimal wiffleball construct were like the ones of BL21(DE3) with the same construct, after the 48h incubation (Figure 8.5 B).

**Conclusion:**

The adjusted incubation time seems to be a game changer for BL21(DE3) as well as for the genome reduced LowMut T7. Before approving this induction protocol, we need to exclude the nature of inclusion bodies by western blot. Due to the limited time, this experiment will be done after the wiki freeze and the data will be included in our presentation at the grand jamboree.

### **Testing our systems in genome-reduced strain (MDS69 LowMutT7)**

**Aim:** Analysing the influence of using genome-reduced strains (MDS69 LowMutT7) as a chassis for our constructs in terms of product yield.

**Experimental setup:** We have done a production assay following protocol #023 with different conditions of genome-reduced strains as well as BL21. We have checked different comparable conditions between our two strains to see how much the reduced genome influences the yield of our pathway. We have used the constructs BL21 + pathway, BL21 + pathway + BMC, MDS69 LowMutT7 + pathway, MDS69 LowMutT7 + pathway + BMC.

**Results:** Unfortunately, we have seen, that transforming our plasmids into genome reduced strains results in lower yields compared to BL21 (see Fig. 8.8). Additionally, we did not see any mentionable production of indigo in the construct MDS69 LowMutT7 + pathway + BMC. We suspect that this is due to the low temperature (18°C) under which we perform protein expression. In different experiments regarding compartmentalization in these genome-reduced strains we have found out that they grow significantly better under higher temperatures. As a result, we would do follow-up experiments comparing different temperature conditions.

![](https://static.igem.wiki/teams/4229/wiki/pictures/results/bmc-1/whatsapp-image-2022-10-13-at-13-35-53.jpeg)

{% endblock %}