iGEM 2023 — Team freiburg

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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# NOTEBOOKS

* Toxin
* B12-Sensing
* Bioproduction

To accomplish all of our ideas and to prove the functionality of our system - **CELLECT**, we decided to split our team into three subteams: the Toxin group, Riboswitch group, and Bioproduction group. All the wet-lab experiments and preliminary results are shown. For a better understanding, a short experimental timetable of each subgroup is provided at the beginning of each PDF.

### Toxin-Notebook

All Toxin group labwork are shown in the following .pdf

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### B12-sensing-Notebook

All B12-Sensing (Riboswitch group) labwork are shown in the following .pdf

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### Bioproduction-Notebook

All Bioproduction group labwork are shown in the following .pdf

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[General protocols](https://static.igem.wiki/teams/4604/wiki/notebook/production-fertig-1.pdf)

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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# ENGINEERING SUCCESS

* Cloning
* Biosensor Riboswitch
* AdoCbl detection
* Toxin Expression

Our goal was to design a self-regulated system to optimize bioproduction and ensure plasmid stability. Therefore, we designed a three component system containing a riboswitch, a toxin-antitoxin system and a gene of interest for production (*bluB*). Each part is highly regulated and yet they interact with each other to form an autoregulatory circuit that regulates the survival of the engineered cells. To achieve our goal, we went through several engineering cycles. The first step of the engineering cycle is designing the project. Followed by the second step, the construction of the system. Third, testing and measuring the results and finally learning from our data to finetune the system until the desired results are reached.

CELLECT is composed of three individual parts and while testing each of them, we went through each of the engineering cycles *multiple times*:

* dealing with our chosen cloning method
* the expression of the toxin, MazF, in *E. coli*
* expression of AdoCbl and
* the design of the riboswitch

# Cloning

Our system contains multiple separate parts. To test their individual functionality and to have the possibility to change specific sequences as ribosome binding sites (RBS) or promoters, we designed multiple plasmids for testing each part individually. Therefore, it was necessary to choose a cloning strategy which allowed such modularity to simplify our cloning process.

**Cycle 1**

![design](https://static.igem.wiki/teams/4604/wiki/eng-cycle/design.png)

After some deliberation, we decided on the Golden Gate method. One big advantage of this method is that we could assemble single and multiple DNA fragments even in the presence of redundant elements. Since no one from our team has had any previous experience in cloning, we wanted to chose a Golden Gate enzyme which, according to research, has a high efficiency. The enzyme we decided on using as a restriction enzyme is BsmBI. Following, we designed overhangs with the corresponding sites to each fragment.

![build](https://static.igem.wiki/teams/4604/wiki/eng-cycle/build.png)

To build our plasmids various fragments were synthesized: Antitoxin, *bluB* and the riboswitch combined with the toxin.
Some of the fragments as the TetR- and Amp promoter, *mTurquoise*, *mVenus* and the rrnB Terminator, we could amplify out of plasmids we got from our PI. After running PCRs to ensure that the restriction sites of BsmBI and the right overhangs were inserted, we cloned our fragments into the pGGAselect backbone.

![test](https://static.igem.wiki/teams/4604/wiki/eng-cycle/test.png)

The DNA construction was amplified and analyzed by agarose gel electrophoresis. It was successful for two of our plasmids containing *bluB* and the toxin. However, we faced multiple problems using this approach. The efficiency of our assemblies was very low, which led us to screen dozens of colonies on our plates in search of one successful clone.

![improve](https://static.igem.wiki/teams/4604/wiki/eng-cycle/improve.png)

We learned that we could adjust the concentration of chloramphenicol on our plates to minimize the amounts of clones that do not carry our target plasmid. We further contacted researchers with experience in Golden Gate cloning to get advice. We learned that the enzyme we had chosen to use for digesting our fragments (BsmBI) seems to be less common than other restriction enzymes used in Golden Gate. In addition, a post-doc (Dr. Morgane Batzenschlager) from a lab next door at the university advised us to use a backbone other than pGGAselect, e.g. EC47732 pL1V-F1-47732 with blue/white selection after addition of X-Gal, AmpR-BsaI or Bpil cloning to facilitate screening of our clones. Furthermore, we learned that incubating the reaction overnight and at lower temperatures and with more cycles could be beneficial. BsmBI works optimal at 55°C but temperatures over 45°C inhibit activity of the T4 Ligase. Even if we lowered the temperature to 42°C, as recommended by NEB, where the T4 Ligase should not be affected, it seemed that BsmBI cut less efficient and therefore this approach did not improve our results. Additionally, we figured out that we could avoid contamination of the original backbone plasmid by doing a DPN1 digest overnight.

![test](https://static.igem.wiki/teams/4604/wiki/eng-cycle/test.png)

However, even by implementing everything we learned, we still didn’t get good results from our cloning strategy. We then cut all our fragments individually and loaded them onto a gel to find out if there were any problems already with the restriction digest by BsmBI.

![improve](https://static.igem.wiki/teams/4604/wiki/eng-cycle/improve.png)

By analyzing our agarose gels, we figured out that digesting our terminator did not work efficiently. It is possible that the random bases we decided on as the overhangs and also as buffer between the recognition and restriction site caused methylation that made it impossible to cut them. Since we had already successfully cloned the riboswitch with Gibson Assembly and our supervisors had the most experience with this cloning method, we decided to switch completely to Gibson Assembly.

![design](https://static.igem.wiki/teams/4604/wiki/eng-cycle/design.png)

By changing our cloning method to Gibson it was necessary to design new primers for each fragment. We paid attention to ensure that the primers had sufficiently long binding sites and that the overhangs did not have a melting temperature under 50°C.

![build](https://static.igem.wiki/teams/4604/wiki/eng-cycle/build.png)

Using the plasmids we already cloned via Golden Gate as a template, we could shorten their sequence via PCR or include fragments in them.

![test](https://static.igem.wiki/teams/4604/wiki/eng-cycle/test.png)

After amplifying each plasmid we did an DPN1 digest overnight instead of 15 min. to ensure that we obtained pure amplified DNA fragments.

![improve](https://static.igem.wiki/teams/4604/wiki/eng-cycle/improve.png)

We learned that we achieved results more efficiently by switching to Gibson Assembly. With this method we cloned most of our plasmids.

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# Biosensor Riboswitch

To test the function of our riboswitch and to have a cost-effective detection method besides the ethanolamine-medium, we decided on designing a biosensor. It was possible to test whether our riboswitch is functional by measuring sfGFP fluorescence. Even though we tested three different riboswitches, this engineering cycle is specific for the sensor containing the *E. coli* K12 riboswitch.

**Cycle 2**

![design](https://static.igem.wiki/teams/4604/wiki/eng-cycle/design.png)

After a long research period, we decided on using the naturally occuring riboswitch from *E. coli* K12, which can detect B12 and differentiate between Cobinamide and Cobalamin[[1]](#cycle_ref1). Our riboswitch negatively affects the expression genes downstream when its ligand adenosylcobalamin (AdoCbl) binds. Therefore, we designed our biosensor so that the riboswitch is located upstream of the *lacI* repressor gene, which in turn regulates the expression of a fluorescent protein, such as the superfolder GFP (sfGFP). When AdoCbl is present, the riboswitch represses LacI expression, the trc promoter in front of *sf*GFP is no longer repressed and sfGFP is produced.
 In other words, our construct reverses the inhibition of the riboswitch, resulting in a positive readout through increased fluorescence at elevated AdoCbl concentrations. The first step was to find a method to test whether the biosensor only expresses sfGFP in the presence of the target compound AdoCbl.

![build](https://static.igem.wiki/teams/4604/wiki/eng-cycle/build.png)

The riboswitch was inserted between the lacI promoter and *lacI*. In order to be able to upload the biosensor as a composite part to the registry of iGEM, a silent mutation would have been necessary between the trc promoter and the *sf*GFP gene to remove an EcoRI restriction site. However, since the reporter gene is variable in this sensor and can be exchanged with any reporter of choice, we decided to keep on working without the silent mutation.
Therefore, sensor [piG\_K12BSa](http://parts.igem.org/Part%3ABBa_K4604026) consists of the lacI promoter, the riboswitch, the *lacI* repressor gene and the trc promoter.

![test](https://static.igem.wiki/teams/4604/wiki/eng-cycle/test.png)

In the experiments we performed to test the biosensor we treated cultures with various concentrations of AdoCbl and measured the fluorescence. We observed a high background fluorescence signal even without AdoCbl supplementation, which implied an insufficient repressor production. Moreover, the total fluorescence signal of the sensor, induced with IPTG, is overall lower than the one of a comparable plasmid containing sfGFP induced with IPTG. This implied that too much repressor was present.
Also the differences in fluorescence with different amounts of AdoCbl added to the medium were minor (see more on [Sensing B12](https://2023.igem.wiki/freiburg/riboswitch-results)).

![improve](https://static.igem.wiki/teams/4604/wiki/eng-cycle/improve.png)

A similar problem was also observed for a toxin experiment. There, insufficient toxin expression was supposedly caused by the RBS which is part of the riboswitch. From literature research we learned that this specific RBS usually controls the expression of a transporter protein that isn't required in high amounts, so we concluded the RBS being rather weak[[2]](#cycle_ref2).
A weak RBS would explain the high fluorescence when the sensor is uninduced. To incorporate the riboswitch to the previously mentioned plasmid we had removed the original RBS of *lacI*. If the RBS of the riboswitch is significantly weaker than the original one, that would explain low repressor production.

![design](https://static.igem.wiki/teams/4604/wiki/eng-cycle/design.png)

We designed new gibson primers with which we could add a supposedly [stronger RBS](http://parts.igem.org/Part%3ABBa_K4604004), taken out of the TetR promoter of plasmid piG\_23, directly between riboswitch and *lacI*, hoping to not disturb the conformation of the riboswitch.

![build](https://static.igem.wiki/teams/4604/wiki/eng-cycle/build.png)

We amplified the backbone via PCR, digested the amplified DNA with DpnI overnight, and inserted the stronger RBS of piG\_23 via Gibson Assembly.

![test](https://static.igem.wiki/teams/4604/wiki/eng-cycle/test.png)

We repeated the whole experiment with the recloned version, [piG\_K12BSb](http://parts.igem.org/Part%3ABBa_K4604027) and noticed that compared to the previous construct, containing the old RBS, the fluorescence overall decreased.

![improve](https://static.igem.wiki/teams/4604/wiki/eng-cycle/improve.png)

We deduced that the high fluorescence in the uninduced state, also not including any AdoCbl, actually was a consequence of the weak RBS provided by the riboswitch. However, this did not explain the minor differences in fluorescence we saw when AdoCbl was added to the medium, nor the overall low fluorescence when we induced the sensor with IPTG.
IPTG is supposed to bind any present repressor, therefore the reduced fluorescence in presence of IPTG suggested either a lower sfGFP production, or an excessive constant repressor expression.
This new insight, certainly unlocked a solution for the issue regarding the toxin that has not been working when the riboswitch was located upstream of the gene. However, it did not improve the function of the sensor past lowering the background fluorescence.
It is still puzzling that the sensor is unable to reach fluorescence as high as the IPTG induced pIG23\_04 plasmid. Because if the only difference had been the weak RBS, an insufficient repressor expression of the *lacI* gene would cause the sensor to always show fluorescence as brightly as the induced original plasmid. As this is obviously not the case, an overproportional repressor production would be the explanation. Thus, even when AdoCbl is present, which is supposed to activate the riboswitch and stop repressor production of LacI, a decreased fluorescence is to be expected.

To find a solution, we firstly need to understand the different ways in which the riboswitch and IPTG inhibit the repressor LacI. While IPTG binds all present repressor, changing its conformation and therefore stopping it from binding and repressing the trc promoter, the riboswitch doesn´t inhibit already present LacI. The riboswitch changes its conformation in the presence of AdoCbl, inhibiting transcription and translation of *lacI*. Simplified, IPTG removes any produced repressor, while the riboswitch only stops production of more repressor. Earlier produced LacI is still present in the system and capable of repressing the trc promoter and consequential sfGFP production. In theory, it could also be possible that the repressor is still produced because not all copies of the riboswitch are always inhibited, or because repressor production starts before AdoCbl can bind to the riboswitch. In any case, some LacI would always be present until it is degraded. This could be the explanation for the minor differences in fluorescence with different AdoCbl concentrations.
As a result, a new solution for improving the detection ability of the sensor would be to change the experimental set up.

![design](https://static.igem.wiki/teams/4604/wiki/eng-cycle/design.png)

A way of testing the repressor production would be measuring sensor activity at multiple time points over an extended period of time. So far measurements were taken only 12 hours after AdoCbl was added to the medium. So theoretically, this timespan could either be too much or too little time to accurately depict the function of the sensor.
If it's too long, too much repressor could have been expressed through leakiness or because all riboswitch can not be activated at all times.
If it is too short, previously produced repressor may not have been degraded yet, and even if at a certain point all riboswitches on all plasmids ware active and able to suppress LacI production, already produced LacI would still be present and able to suppress sfGFP production.

![test](https://static.igem.wiki/teams/4604/wiki/eng-cycle/test.png)

So far we were unable to test the sensor in this new experimental set up.

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# AdoCbl Detection

In order to evaluate whether LB medium contains significant amounts of B12 (which would consequently exclude its use in further experiments) and to prove that our *E. coli* cells actually synthesize AdoCbl, we searched for a method to detect B12, more specifically, AdoCbl.

**Cycle 3**

![design](https://static.igem.wiki/teams/4604/wiki/eng-cycle/design.png)

The first B12 detection method we chose to test was ELISA, using a Vitamin B12 ELISA kit (DEB12E01) from Demeditec. It was a generally well-documented detection method, quicker and easier compared to LC-MS, and we could perform it in our own lab. Important considerations that influenced our choice. However, finding an ELISA designed for B12 detection in cell lysates specifically remained a challenge. The Demeditec kit we tested was designed for cyanocobalamin detection in vitaminized foods, as the company contact person made us aware. At the same time, they also shared with us a publication documenting the same ELISA kit being used for B12 detection in cell lysates of *Mycolicibacterium smegmatis*[[3]](#cycle_ref3). Therefore, we finally decided to test the applicability of the kit with *E. coli* cell lysates and LB medium.

![build-test](https://static.igem.wiki/teams/4604/wiki/eng-cycle/build-test.png)

First, we used the ELISA kit to test B12 concentration in LB medium. Additionally, we tested two other media used for *E. coli* and cyanobacteria cultivation (both supposedly containing no B12), and also cell lysates with different amounts of AdoCbl added (as a validation of our detection method). It was important to test the kit’s responsiveness to varying amounts of AdoCbl in cell lysates (and its usability with cell lysates in general) since that would determine its usability for our project. Lastly, we tested the kit’s specificity with samples containing cobinamide, the precursor we would add exogenously to the medium for B12 production.

![improve](https://static.igem.wiki/teams/4604/wiki/eng-cycle/improve.png)

A high B12 content was detected in the LB medium and therefore we decided to change our cultivation medium for B12 production experiments to M9 (minimal) medium. However, we also had to change the detection method for further experiments since the kit appeared to have a high cross-reactivity with cobinamide (which would always be present in the production cultures). Next, we realized that converting all cobalamin forms in cell lysates to cyanocobalamin (the B12 form the specific ELISA kit is made for) is not possible for routine measurements in our lab, and if we converted the photo-labile forms (adenosylcobalamin and methylcobalamin that we expect to produce) to hydroxocobalamin, the kit wouldn't allow for precise detection of the latter. Hence, a different B12 detection method was needed.

![design](https://static.igem.wiki/teams/4604/wiki/eng-cycle/design.png)

As an alternative we have chosen ethanolamine medium as an easy and cheap detection method. The principle behind the ethanolamine medium is that it only allows bacterial growth when AdoCbl is present. *E. coli* produce ethanolamine ammonia lyase (EAL), an enzyme that breaks down ethanolamine into ammonia and acetaldehyde. Furthermore, EAL requires AdoCbl as a cofactor. The fact that *E. coli* can utilize ethanolamine as a nitrogen source only in the presence of AdoCbl, the ethanolamine medium offers a convincing detection method[[4]](#cycle_ref4).

![build](https://static.igem.wiki/teams/4604/wiki/eng-cycle/build.png)

After some unsuccessful attempts at making the actual ethanolamine medium, we finally found a protocol which worked.

![test](https://static.igem.wiki/teams/4604/wiki/eng-cycle/test.png)

The aim of our first experiment was to test whether *E. coli* is able to grow in the medium with ethanolamine as the sole nitrogen source, both with and without AdoCbl supplementation. We did a growth curve comparison for LB medium and Ethanolamine medium. We furthermore tried to use the differences in growth of bacteria supplemented with different amounts of AdoCbl to create a calibration curve.

![improve](https://static.igem.wiki/teams/4604/wiki/eng-cycle/improve.png)

We learned that with this method it is not possible to determine precise AdoCbl concentrations, only qualitatively. However as previously published, it is an efficient and easy method for qualitatively measuring if B12 was present. However, we still had the wish to determine the exact concentration of B12 and other precursors in order to prove actual B12 production. We learned that mass spectrometry can be used to determine more precisely how much AdoCbl is present in the cells and whether it really is AdoCbl or another substance. We ended up coming in contact with a research group at the University Medical Center of Freiburg who was willing to do these measurements for us. We are incredibly grateful for their support!

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# Toxin Expression

For the functionality of our system a potent toxin is essential. We performed experiments for toxin expression, attempting to assess the lethality of MazF. Unfortunately, the first results implied that our chosen toxin was unexpectedly non lethal to the cells. Subsequently, we searched for the cause of inadequate toxicity.

**Cycle 4**

![design](https://static.igem.wiki/teams/4604/wiki/eng-cycle/design.png)

To test toxin expression, we designed a plasmid containing the toxin gene *mazF* downstream of the riboswitch, including the associated RBS. We chose to test the toxin together with the riboswitch to directly determine the expression level of the toxin that would eventually be present in our self-regulating system. We added a 6xHis-Tag to the toxin to perform western blot experiments to visualize toxin expression.

![build](https://static.igem.wiki/teams/4604/wiki/eng-cycle/build.png)

We took the toxin sequence, as well as the riboswitch sequence, from a publication[[5]](#cycle_ref5)[6] and synthesized it as a contiguous sequence. The fragment was then amplified by PCR, cloned into pGGAselect via Gibson assembly and finally transformed into *E. coli* MG1655.

![test](https://static.igem.wiki/teams/4604/wiki/eng-cycle/test.png)

To test the toxicity of expressed *mazF*, we measured optical density and determined colony forming units (CFUs) for different doxycycline (DOX) inducer concentrations. We expected to see reduced growth (decrease in optical density), as well as decreasing CFUs. Unfortunately, we did not see a decrease in survival with higher concentration of inducer. Even at a DOX concentration of 1000 ng/mL, at which the toxin should have been sufficiently expressed, no major changes were observed compared to the negative control. However, western blot results showed toxin expression after DOX induction.

![improve](https://static.igem.wiki/teams/4604/wiki/eng-cycle/improve.png)

We found that our toxin was expressed with an upstream riboswitch, but was not inducing cell death. We had become aware that we might get into trouble with the toxin already early on in the project. We introduced our idea at the biology [Faculty Day](https://www.bio.uni-freiburg.de/events/20230630-fakultaetstag-en) of the university on June 30th, 2023 (see more on [integrated human practise](https://2023.igem.wiki/freiburg/human-practices)). It is an event for all those interested in biology, and where the various projects of the university's research groups are presented. The question arose to what extent we prevent the cells from mutating the toxin, or the associated promoter, over time. Rationally, bacteria in our cultures which no longer express a functional MazF would gain an advantage, and would therfore prevail. Since our toxin-antitoxin genes do not overlap, as they do in the native system, the cells are not forced to keep the functional *mazF* gene[[7]](#cycle_ref7).

![design](https://static.igem.wiki/teams/4604/wiki/eng-cycle/design.png)

We redesigned the system, so that the bacteria gain a survival advantage when no mutattions occur in the promoter regulating toxin expression. We had the idea to implement a second antibiotic resistance e.g. kanamycin behind the TetR promoter and between the *bluB* gene and riboswitch and toxin. If the bacteria mutated the TetR promoter, they also lose the ability to express the kanamycin resistance and will not be able to survive the selection pressure(see more on [design page](https://2023.igem.wiki/freiburg/design)).

We redesigned the system, so that the bacteria have a survival advantage when they do not mutate the promoter regulating toxin expression. We had the idea to implement a second antibiotic resistance e.g. Kanamycin behind the TetR promoter and between bluB gene and riboswitch and toxin. If the bacteria mutate the TetR promoter they also lose the expression of Kanamycin and will not be able to survive the selection pressure (see more on the design page).

We also decided to redesign the protocol of our experiment. For the next experiments we collected single colonies from the CFUs and sent them for sequencing to gain more information about the possible mutation of the toxin.

![test](https://static.igem.wiki/teams/4604/wiki/eng-cycle/test.png)

After several cloning cycles we did not manage to clone the kanamycin plasmid, so we decided to focus on cloning and testing the individual parts.
We screened several generations of bacteria, which had been induced in our experiments to express the toxin, by sequencing. Primers were used which specifically allow sequencing of the toxin.

![improve](https://static.igem.wiki/teams/4604/wiki/eng-cycle/improve.png)

According to the sequencing results of the toxin, we had no mutations inside the relevant gene which could have affected the function of the toxin. Apparently, the concerns of toxin mutation do not seem to have any impact on our system in the period we have tested. However, despite this positive finding the problem remained, that the toxin didn’t cause cell death upon induction.

After long troubleshooting, we looked for possible answers as to why our toxin experiments were unsuccessful. We hypothesized that the RBS of the riboswitch might be too weak to produce enough toxin to induce cell death or inhibit cell growth. After extensive literature research,this conclusion was further supported[[8]](#cycle_ref8).

. Thus, we designed a new plasmid containing the toxin with a different, stronger RBS. In order to create similar conditions, this plasmid, like the previous toxin plasmid, also contains the TetR promoter, the toxin and the rrnB terminator. The RBS used, originates from the TetR promoter region.

![build](https://static.igem.wiki/teams/4604/wiki/eng-cycle/build.png)

As a backbone, we used a plasmid that we received from our supervisor. This already contained the TetR promoter and the rrnB terminator. We amplified our toxin from the previous plasmid and inserted it into the backbone via Gibson Assembly. The final plasmid (piG\_23) was transformed into *E. coli* MG1655.

![test](https://static.igem.wiki/teams/4604/wiki/eng-cycle/test.png)

For simplicity, the experiment with piG\_23 and pGGAselect as a control, could be carried out in LB medium due to the lack of a riboswitch. Our results showed a decrease in growth and CFUs following induction with 50 ng/mL DOX.

![improve](https://static.igem.wiki/teams/4604/wiki/eng-cycle/improve.png)

This was a first indication of increased toxin expression and consequently increased toxin activity in the cells. We suspect that the RBS of the riboswitch was indeed the cause of the previously insufficient toxin activity.

![design](https://static.igem.wiki/teams/4604/wiki/eng-cycle/design.png)

To make sure that our assumptions were correct, we cloned new toxin constructs that contain the different RBS. Thus, a plasmid (piG\_23b) was designed with the weaker RBS originating from the riboswitch . In this case, we would expect the toxicity to decrease and the levels to match those of the other toxin plasmid (with riboswitch).

![build](https://static.igem.wiki/teams/4604/wiki/eng-cycle/build.png)

New primers were designed to exchange the respective RBS via Gibson Assembly and to reintroduce them into the backbone. The cloned plasmids were then transformed into *E. coli* MG1655. We added a 6xHis-Tag to the toxin to perform western blot experiments to visualize toxin expression.

![test](https://static.igem.wiki/teams/4604/wiki/eng-cycle/test.png)

Cells containing piG\_23b, as well as control cells containing pGGAselect, showed a steady increase in growth over time in the non-induced and induced state with 100 ng/mL DOX(see more on [toxin/antitoxin results page](https://2023.igem.wiki/freiburg/toxin-antitoxin-results)). This result indicates no difference in growth, while significant growth inhibition can be observed for piG\_23 cells induced with 100 ng/mL DOX.

![improve](https://static.igem.wiki/teams/4604/wiki/eng-cycle/improve.png)

We learned that by replacing the RBS of the riboswitch with a stronger RBS, the expression of downstream genes is altered.

![build](https://static.igem.wiki/teams/4604/wiki/eng-cycle/build.png)

Next we would be interested to clone the TetR RBS into the plasmid consistin of TetR/Riboswitch/Toxin/Terminator in addition to the already existing Riboswitch RBS. This plasmid thus contains two RBS in order to guarantee the functionality of the riboswitch. In an experiment with this plasmid, we expect an increased toxicity, similar to piG\_23. Unfortunately this was out of the capabilities of our iGEM project.

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# References

* [1]Cai Y, Xia M, Dong H, Qian Y, Zhang TC, Zhu B, et al. Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti. 2018 May 11;18(1).
* [2] Li GW, Burkhardt D, Gross C, Weissman Jonathan S. Quantifying Absolute Protein Synthesis Rates Reveals Principles Underlying Allocation of Cellular Resources. Cell. 2014 Apr;157(3):624–35
* Li GW, Burkhardt D, Gross C, Weissman Jonathan S. Quantifying Absolute Protein Synthesis Rates Reveals Principles Underlying Allocation of Cellular Resources. Cell. 2014 Apr;157(3):624–35[3]
* [4] CHANG GW, CHANG JT. Evidence for the B12-dependent enzyme ethanolamine deaminase in Salmonella. Nature. 1975 Mar;254(5496):150–1.
* [5]Using a Riboswitch Sensor to Examine Coenzyme B12 Metabolism and Transport in *E. coli*. Chemistry & Biology [Internet]. 2010 Jul 30 [cited 2021 Apr 5];17(7):756–65. Available from: https://www.sciencedirect.com/science/article/pii/S1074552110002437?via%3Dihub
* [6]Escherichia coli str. K-12 substr. MG1655, complete genome. NCBI Nucleotide [Internet]. 2022 Mar 9 [cited 2023 Oct 9]; Available from: https://www.ncbi.nlm.nih.gov/nuccore/NC\_000913.3?report=fasta&from=2910756&to=2911091&strand=true
* [7]Lemos JAC, Brown TA, Abranches J, Burne RA. Characteristics ofStreptococcus mutansstrains lacking the MazEF and RelBE toxinâantitoxin modules. FEMS Microbiology Letters. 2005 Dec;253(2):251–7.
* [8]Li GW, Burkhardt D, Gross C, Weissman Jonathan S. Quantifying Absolute Protein Synthesis Rates Reveals Principles Underlying Allocation of Cellular Resources. Cell. 2014 Apr;157(3):624–35.

{% endblock %}

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

# GENERAL PROTOCOLS

* Media
* Antibiotic-Stocks
* Cloning and Transformation
* SDS and Western-Blot
* Microscopy
* Buffers

During the progress of iGEM, we had many different experimental setups to test and research all of our individual parts. For reocurring procedures, we collected and adapted many general protocols for various daily lab tasks. On this page, all general protocols are listed. These protocols were improved by us to fit our needs and setups and were used in all aspects of our lab work.

### Media Protocols

Standard protocols to create media and making the corresponding plates

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[General protocols Mediums](https://static.igem.wiki/teams/4604/wiki/gen-protocols/medium.pdf)

### Antibiotic-Stocks

Standard protocols for antibiotic-stocks and storage conditions

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[General protocols](https://static.igem.wiki/teams/4604/wiki/gen-protocols/antibiotic-stock.pdf)

### Cloning and Transformation Protocols

Standard protocols for each different cloning, transformation, PCR-set up and DNA preparation

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[General protocols](https://static.igem.wiki/teams/4604/wiki/gen-protocols/cloning-protocols.pdf)

### SDS and Western-Blot

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[General protocols](https://static.igem.wiki/teams/4604/wiki/gen-protocols/sds-gel-and-western-blot.pdf)

### Microscopy

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[General protocols](https://static.igem.wiki/teams/4604/wiki/gen-protocols/microscopy.pdf)

### Buffer Protocols

Standard protocols for buffer preparation

---

[General protocols](https://static.igem.wiki/teams/4604/wiki/gen-protocols/buffers.pdf)

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# COMPOSITE SYSTEM

* Combining All Parts
* Inducible Kanamycin Resistance
* Outlook for CELLECT

## Testing the Functionality of piG\_10b for B12 Bioproduction

### Aim

In this experiment we tested the functionality of piG\_10b, the second prototype of CELLECT, in a B12 production setup and compared it with piG\_10a, piG\_01b and pGGAselect as a negative control. We investigated cell growth, B12 yield and expression of *bluB* and *mazF*.

### Experimental setup

| Name | Short description | BioBrick |
| --- | --- | --- |
| piG\_01b | pTetR\_bluB | [BBa\_K4604015](http://parts.igem.org/Part%3ABBa_K4604015) |
| piG\_10a | tetR\_bluB\_riboK12\_mazF | [BBa\_K4604021](http://parts.igem.org/Part%3ABBa_K4604021) |
| piG\_10b | pTetR\_bluB\_RiboK12\_tetRBS\_Toxin | [BBa\_K4604022](http://parts.igem.org/Part%3ABBa_K4604022) |

**Figure 1: Table explaining all plasmids used in this experiment.**  All information regarding the cloning of these plasmids is available on our [Cloning plasmids](https://2023.igem.wiki/freiburg/plasmids) page.

*E. coli* MG1655[piG\_10b], *E. coli* MG1655[piG\_10a], *E. coli* MG1655[piG\_01b] and *E. coli* MG1655[pGGAselect] cells from a glycerol stock were used to inoculate respective 100 mL pre-cultures in M9 medium.

These pre-cultures then grew to sufficient optical densities to inoculate 50 mL cultures to an OD600 of 0.3. Substrates and inducer were added at OD600~0.6. We tested induced and non-induced setups with cobinamide (Cbi) and hydroxocobalamin (OHCbl) for piG\_10b, piG\_10a, piG\_01b and pGGAselect. pGGAselect is the empty backbone to the other 3 plasmids.

The cultures grew at 37°C, shaking at 200 rpm. Optical density was measured every 6 hours during the first 24 hours and once more after 48 hours. Samples for Western Blot were taken after 0, 12, 24 and 48 hours. LC-MS samples were taken 0, 24 and 48 hours after induction and substrate supplementation.

For detection of B12 concentrations with Liquid Chromatography - Mass Spectrometry (LC-MS), we took 1 mL of cell culture, centrifuged it, discarded the supernatant and stored the cell pellet at -80°C. These samples were then purified and exposed to light deliberately before sending for external LC-MS analysis, blinded to prevent bias.
The experiment was repeated a total of two times.

![](https://static.igem.wiki/teams/4604/wiki/composite-results/compf1.png)

**Figure 2: Scheme of the experimental procedure.** Experimental setups contained indicated concentrations of doxycycline (DOX) / Cbi / OHCbl. Cultures were investigated utilizing OD600 and LC-MS measurements as well as SDS-PAGE/Western Blot.

### Results

The setups of *E. coli* MG1655[piG\_10b] induced with doxycycline (DOX) showed no growth, similarly to the culture of *E. coli* MG1655[piGGAselect] treated with Cbi and DOX (Figure 3).
All other cultures grew relatively similar.

![](https://static.igem.wiki/teams/4604/wiki/composite-results/compf2a.png)

![](https://static.igem.wiki/teams/4604/wiki/composite-results/compf2b.png)

**Figure 3: Growth assay of *E. coli* MG1655 in different conditions.** Cultures were induced with DOX and supplemented with the indicated concentrations of Cbi / AdoCbl at an OD600 of ~0.6. (A) Growth curve (B) Bar chart comparison between optical densities after 0 hours and 48 hours. Optical density of each culture was measured using a ThermoScientific NanoDrop 2000c Spectrophotometer. Sample size n=2.

Western Blot shows *bluB* expression only for induced piG\_10a and piG\_01b (Figure 4). No signal for BluB was observed in any of the induced *E. coli* MG1655[piG\_10b] setups.
We did see expression of *mazF* for induced piG\_10b, while none was visible for induced piG\_10a. Additionally, the setup of induced *E. coli* MG1655[pGGAselect] with Cbi displayed a signal for MazF as well.

![](https://static.igem.wiki/teams/4604/wiki/composite-results/compf3a.png)

![](https://static.igem.wiki/teams/4604/wiki/composite-results/compf3b.png)

**Figure 4: BluB and MazF expression in MG1655[piG\_10b] with different substrates, induced and uninduced.** Detection of recombinantly expressed, his-tagged BluB and FLAG-tagged MazF enzymes with SDS-PAGE followed by Western Blot. Detection of the BluB protein was performed with an anti-his antibody. MazF was detected using an anti-FLAG antibody. Loading control: RNA polymerase β-subunit.

We can see B12 production in *E. coli* MG1655[piG\_10a] and *E. coli* MG1655[pGGAselect], induced with DOX and supplemented with Cbi respectively. Induced *E. coli* MG1655[piG\_10a] with Cbi show OHCbl contents about 50% compared to *E. coli* MG1655[piG\_01b] under the same conditions.

![](https://static.igem.wiki/teams/4604/wiki/composite-results/compf4.png)

**Figure 5: OHCbl and contents in modified *E. coli* MG1655 cells from production cultures, measured by LC-MS.** Amounts of OHCbl in g/g DCW were calculated with the concentrations measured in LC-MS samples and DCW averages from a prior experiment correlated to the respective optical densities. LC-MS performed at Hannibal Lab, University MEdical Center Freiburg. Sample size n=2.

### Conclusion

Expression of *bluB* and conversely B12 production does not work in *E. coli* MG1655[piG\_10b]. We observed *mazF* expression and growth inhibition of induced cells cultures.

The results from this experiment imply that neither riboswitch nor *bluB* gene are working in this construct. This is surprising because bacteria carrying piG\_10a do express BluB and produce B12 in the very same experiment. We modified piG\_10a by exchanging the ribosome binding site (RBS) upstream of *mazF* for a stronger RBS, creating piG\_10b. More about the cloning procedures is listed on our [Cloning Plasmids](https://2023.igem.wiki/freiburg/plasmids) page.

As the results from this experiment appeared suspicious to us, we had a look at the sequencing results. The quality of the peaks that would determine single nucleotides was rather low, indicating that there might have been a subculture in the glycerol stock we took the cells from. Unfortunately, we did not manage to repeat this experiment with newly cloned bacteria in time to validate the findings from this experiment.

## Testing Inducible Kanamycin Resistance

### Aim

To test if the Kanamycin resistance on piG\_11 is inducible by DOX and can be further used for the improved design of our system.

### Experimental Setup

*E. coli* MG1655 containing the piG\_11 where incubated in LB at 37°C and 200 RPM for 1 hour. Afterwards they were split into four different setups.

The setups were 2x LB without anything added, LB with 100 ng/mL DOX and LB with 100 ng/mL DOX and 200 nM AdoCbl added.

They were further incubated in their respective setup at 37°C and 200 RPM for 1 hour. Then plated out on LB plates with different Antibiotics added. Antibiotics added to the LB plates were: 3x 50 mg/mL kanamycin and 34 µg/mL chloramphenicol and one plate with just 34 µg/mL chloramphenicol .

![](https://static.igem.wiki/teams/4604/wiki/composite-results/kan-exp.png)

**Figure 6: Scheme of the experimental procedure.**

### Results

We see a lot of bacteria growth for all four plates (Figure 7). But the most colonies can be seen on plate and 4, while individual colonies can be seen on plate 2 and 3.

![](https://static.igem.wiki/teams/4604/wiki/composite-results/kanamycin-platten.png)

**Figure 7: *E. coli* MG1655 containing the piG\_11 plasmid on different antibiotics and induced with AdoCbl and DOX** 1) non induced on a LB with 50 ng/mL kanamycin and 34 ng/mL chloramphenicol plate. 2) induced with 100 ng/mL DOX on a LB with 50 ng/mL kanamycin and 34 ng/mL chloramphenicol plate. 3) induced with 100 ng/mL DOX as well as 200 nM AdoCbl on a LB with 50 ng/mL kanamycin and 34 ng/mL chloramphenicol plate. 4) non induced on a LB with 34 ng/mL chloramphenicol plate.

### Conclusion

The results are inconclusive, because we see the most colonies for the non induced plates.
We would expect a lot of colonies for setup 4) (Figure 7.4) since piG\_11 has CHL resistance as its main antibiotic on the plasmid.
For setup 1) we would expect no colonies since the kanamycin resistance is uninduced and they are grown on kanamycin. This is however not what we observe leading us to conclude that the leaky promoter is leaky enough to support growth on kanamycin (Figure 7.1).

Interesting is that for the setups induced with DOX we observe a lower colony number than for the non induced setups (Figure 7.2 & 7.3). The main reason for this could be the induced toxin even though our early toxin results, comparable on our [Toxin-Antitoxin Results](https://2023.igem.wiki/freiburg/toxin-antitoxin-results) page, have shown that the RBS of the ribsowitch should not support cell death.

This is further supported by the setup with AdoCbl added (Figure 7.3) as we would expect to have more colonies there if the toxin would indead be the cause for the lower colony number. Since AdoCbl should bind the ribowitch leading to lower toxin amounts.

Conclusivly can be said that the kanamycin resistance is not inducable since the leakyness causes the cells to survive without inducing the resistance. Therefore we dit not go forward with this approach. It remains to be seen however if the kanamycin would meet its purpose of protecting the toxin promoter from mutations.

The fact that the cells are kanamycin resistant without induction could even be seen as an advantage since no inducer is requiered and the purpose of protecting the toxin promoter would still be given.

## Outlook for CELLECT

Since we designed CELLECT, our self-regulated-system, to ensure genetic stability and improve bioproduction, the optimal next step would be to integrate it directly into the bacterial genome to eliminate the need of antibiotic resistance. Especially, as large-scale bioproduction does not allow for supplementation of antibiotics due to economical and environmental reasons [[1]](#com_ref1). This was also pointed out by the industrial biotechnology company BRAIN Biotech during our meeting. More about our meeting with BRAIN Biotech is accessible on our [Integrated Human-Practices](https://2023.igem.wiki/freiburg/human-practices) page. However, implementing the system into the bacterial genome would go beyond the scope of our project, so we decided on using plasmids carrying our system as proof of principle.

Unfortunately, due to the shortage of time and the long cloning process, it was not possible to achieve all of our goals. For some parts, we have not yet had the chance to perform the necessary experiments to provide us with the desired answers. So here are some improvements we will implement or tests if we choose to move on with CELLECT, exceeding the iGEM competition.

### Improving Toxin/Antitoxin System

One of the next steps would be to gain more information about the Toxin/Antitoxin interactions by using the fluorescence resonance energy transfer (FRET) method. FRET is a distance-dependent physical process by which energy is transferred from an excited donor fluorophore (mTurquoise) to another acceptor fluorophore (mVenus), when they are in close proximity (10-100Å) to each other [[2]](#com_ref2). By coupling mTurquoise to the toxin *mazF* and fusing the antitoxin mazE to mVenus, we are interested in detecting the formation and the lifespan of the unstable toxin-antitoxin complex.

As toxin expression is regulated through the same inducible promoter as the *bluB* gene, the expression of both proteins is coupled to each other. Therefore the required amount of antitoxin to buffer toxin production after addition of inducer is a crucial question to be answered.
It is essential to determine the lethal toxin concentration in the cell in order to adjust the production of antitoxin accordingly with the help of a constitutive promoter. Ensuring the survival of the cell at the onset of production, preventing cells to die because toxin production is faster than B12 synthesis, while also ensuring cell death in case of unproductiveness.

At the beginning of the project, we looked into several different toxin-antitoxin systems. After intense research and review of the current literature, we decided on MazE/F as most suitable for the moment. It is well studied and has already been implemented as a part of the iGEM registry.
By collecting experimental data on this TAS we want to solidify our choice as the best possibility for CELLECT. If our experimental data where to suggest MazE/F is not suitable we wouldn't hesitate to switch the TAS.
According to a publication, growth inhibitory effects of MazF seem to be higher during the lag phase of a culture compared to mid-log phase cultures of *E. coli* [[3]](#com_ref3). Therefore, we are interested in the effect of DOX induction at OD600=0.1 instead of OD600 = 0.5/6 on MazF toxicity.

### Eliminating Mutation Risks

The most susceptible sites for mutations in our CELLECT plasmid are the toxin gene *mazF* and the TetR promoter in front of *bluB* and *mazF*. As the cells are exposed to a selection pressure favoring random mutations that stop production of the toxin, new subpopulations with mutations of these plasmid parts could emerge.

With sequencing results repeated over a time span of around four days, we were able to show that the toxin gene did not mutate which could have affected the function of the toxin. All details regarding this experiment are obtainable on our [Toxin-Antitoxin Results](https://2023.igem.wiki/freiburg/toxin-antitoxin-results) page.

Even if the concerns of toxin mutation do not seem to have any impact on our system in the period we have tested, we are interested in finding a solution to minimize the probability of mutations that could affect our toxin long term. Implementing CELLECT in a bioreactor would require solid stability for long periods of time and numerous cell generations.

Therefore, we would introduce a second selection marker. As an example, placing a kanamycin resistance gene in front of the riboswitch followed by the toxin under the same promoter. This would be our CELLECT system two. To see more about CELLECT system two, have a look at our [Design](https://2023.igem.wiki/freiburg/design) page.
In theory, if the TetR promoter in front of the riboswitch and toxin gene mutates and therefore transcription of this part is stopped, the kanamycin resistance is also not expressed and the cells are vulnerable to antibiotic selection.
However, the promoter we used for the CELLECT system appeared to be leaky. Therefore during initial experiments with the kanamycin resistance, we experienced that even without induction via DOX cells survived in media supplemented with kanamycin. The solution would be to replace the leaky TetR promoter with a tighter inducible promoter for CELLECT system two. Optionally, a higher kanamycin concentration could be used to counteract the leaky expression, as induction still increases expression of the resistance gene.

Despite having these options to improve CELLECT in mind, time was figuratively slipping through our fingers. Therefore we decided to focus on our system without the kanamycin resistance firstly.

A second idea to stabilize the mutation-sensitive areas is an overlap in the toxin and antitoxin gene. Creating artificial overlaps and frameshifts is a technique developed specifically for improved gene stability [[4]](#com_ref4). For an improved CELLECT system, the toxin and antitoxin genes could be arranged with an overlap including a shifted frame, causing mutations in the toxin gene to affect the antitoxin gene as well. As the toxin is more stable than the antitoxin, such a mutation would be deadly for the cell. Based on this, we thought of adding a second antitoxin gene in a frame shift to the toxin gene, protecting the toxin from mutations. This method, however, requires a lot of know-how and optimisation and was therefore not feasible to pursue during iGEM.

### Improving Production

Due to the fact that hardly any data exist on whether BluB differs in its activity in the monomer and in the dimer, we decided to model this using biological structural analysis and ultimately molecular dynamics simulations. All results and data generated with this model of molecular dynamics is provided on our [Modeling](https://2023.igem.wiki/freiburg/model) page.

To do this, we first used AlphaFold [[5]](#com_ref5), a tool for protein structure prediction based on an amino acid sequence. BluB as a monomer and as a fusion dimer were created from our plasmid sequence of piG\_01. We showed that our artificial fusion dimer is stable in our simulation of over 30 ns and forms the enzymatic pocket in a similar way as published crystallographic BluB data [[6]](#com_ref6). The BluB monomer however showed imperfect protein folding because the c-terminal tails of BluB stabilize the substrate Flavin mononucleotide.

We therefore suggest that a fusion BluB could help improve AdoCbl production yields because enzymatic BluB is bound to its complex partner, increasing the likelihood of the dimer forming and overall stability. We plan on cloning this fusion protein and are thrilled to see if we can achieve experimental approval of this assumption by reaching higher B12 yields.

### Improving Riboswitch

For the implemented riboswitch, we were sadly unsuccessful in constructing and cloning a reliable and precise AdoCbl sensor. Although different AdoCbl concentrations only lead to minor, yet expected changes in fluorescence, supplementing Cbi does not show any predictable effect on fluorescence, thereby suggesting that the riboswitch does fold accordingly. You can find more details regarding the riboswitch on our [Sensing B12 Results](https://2023.igem.wiki/freiburg/riboswitch-results) page.

However, at this point, we are unfortunately still unsure whether the riboswitch we have chosen folds as intended, when another gene is expressed upstream.

The limitation for the need of an already existing riboswitch could possibly be overcome by the use of a synthetic riboswitch made on the basis of an aptamer [[7]](#com_ref7). However, this is a complex process and requires extensive testing which was outside of the capabilities of our iGEM project.

### Implementing Missing Controls

Due to time pressure we currently do not have a negative control for our system. This would be a mutated, non-functional *bluB*. Here, no vitamin B12 is produced and the cells should therefore die due to lacking inhibition of the toxin gene via the riboswitch. Such a plasmid (tetR\_mut\_bluB\_riboK12\_mazF) is already designed, but so far has not been cloned successfully.

### Alternative Applications

A fascinating next step for the development of CELLECT would be exploring alternative applications aside from bioproduction. With its universal design, the autoregulatory system is not limited to bioproduction only. It can also be used for 1) degradation by making substance-degrading microbes dependent on the associated intermediate and for 2) biocontainment: in this case, the used organisms survive just as long as the intermediate is produced from the degraded product.

# References

* [1]Wang ZK, Gong J, Qin J, Li H, Lü ZM, Shi J, et al. Improving the intensity of integrated expression for microbial production. ACS Synthetic Biology [Internet]. 2021 Nov 5;10(11):2796–807. Available from: https://doi.org/10.1021/acssynbio.1c00334
* [2]Sekar RB, Periasamy A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol. 2003 Mar 3;160(5):629-33. doi: 10.1083/jcb.200210140. PMID: 12615908; PMCID: PMC2173363. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2173363/
* [3]Potnis AA, Raghavan PS, Shelke A, Nikam TD, Rajaram H. Comparative analysis of MazEF and HicAB toxin–antitoxin systems of the cyanobacterium,Anabaena sp. PCC7120. Visweswariah S, editor. FEMS Microbiology Letters. 2016 Dec 8;364(1):fnw279.* [4]Decrulle AL, Frénoy A, Meiller-Legrand TA, Bernheim A, Lotton C, Gutierrez A, et al. Engineering gene overlaps to sustain genetic constructs in vivo. PLoS computational biology [Internet]. 2021 Oct 1 [cited 2023 Oct 9];17(10):e1009475. Available from: https://pubmed.ncbi.nlm.nih.gov/34624014/
  * [5] Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021 Jul 15;596(7873):583–9.
  * [6]PDB ID: 2ISJ
  * [7]artificial riboswitch Findeiß S, Etzel M, Will S, Mörl M, Stadler P. Design of Artificial Riboswitches as Biosensors. Sensors [Internet]. 2017 Aug 30;17(9):1990. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621056/

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