Difference between revisions of "Part:BBa K4604023"

Latest revision as of 14:18, 12 October 2023

piG_21 (tetR_bluB_riboK12_mazF_AmpProm_mazE)

BioBrick piG_21 is a unit of expression consisting of the tet promoter (consisting of the tet repressor (BBa_K4604002) and tet operator (BBa_K4229059), bluB (BBa_K4604005), an AdoCbl riboswitch (BBa_K4604031), mazF (toxin, BBa_K4604011), the rrnB terminator (BBa_K4604003), the constitutive amp promoter, (BBa_K2308010), mazE (antitoxin, BBa_K4604037) and the rrnB terminator (BBa_K4604036). The backbone we used in most experiments is pGGAselect [BBa_K3841023], a plasmid commonly used with Golden Gate cloning that has a chloramphenicol resistance.

Notice

This BioBrick was uploaded since this system is what CELLECT is about, we see it as our best composite part. Due to time constraints, we did not manage to test the parts combined, only separately and in silico. Therefore, keep in mind that the result presented on this page did not arise from testing this exact setup. Despite that, we still want to present our results that combined make up this composite part and for it to be displayed in a coherent way. Partial results are also uploaded onto the respective registry pages.

Usage and Biology

Antibiotic resistances dominate as a comfortable selection marker and play a crucial role in genetic engineering overall, ensuring the heterologous plasmid is retained in the organism of choice. Intriguingly, plasmid retention does not guarantee plasmid encoded gene expression, since bacteria can use methods such as methylation or deletion to withdraw from the metabolic burden [1]. This means that bacteria have their ways of not expressing inserted genes, defying the researchers control over their constructs. This composite BioBrick (BBa_K4604023) represents a novel tool to ensure not just the presence of a plasmid but also the expression of recombinant genes on it in bacteria. The key idea is to couple the production of a metabolite to the survival of the cell and thereby making it autoregulatory.

Modularity

Our composite part incorporates an Adenosylcobalamin (AdoCbl) riboswitch (BBa_K4604031), a toxin-antitoxin system (BBa_K4604037/BBa_K4604011) and an enzyme, bluB (BBa_K4604005) needed to boost the AdoCbl production in E. coli (Fig. 1). The system was tested and developed with AdoCbl production as a proof of principle, allowing only AdoCbl producing cells survive. To design an effective system for another compound, the riboswitch has to be changed to be able to sense the desired compound and a single gene or pathway that produces the desired compound. Consequently, the system is conveniently adjustable for any desired compound detectable by a riboswitch. 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 [2]. Synthetic aptamers can be generated through in vitro systematic evolution of ligands by exponential enrichment (SELEX) which allows for an even broader field of application of the composite part. However, this is a complex process and requires extensive testing outside the scope of our iGEM project. The bluB gene can similarly be exchanged with an enzyme that fits the chosen product. The host organism is also variable; even though the BioBrick is adapted to use in E. coli, with adjustments of the promoter/terminator region, the toxin-antitoxin system, as well as the origin of replication it could be applicable to any chassis organism.

AdoCbl production

We decided on the AdoCbl (a bioavailable form of vitamin B12) production as a proof of concept. Vitamin B12 is an essential nutrient, humans are dependent on for the production of red blood cells, the synthesis of the DNA and the function of nerves. To form the complete AdoCbl synthesis pathway in E. coli, it would require 28 additional genes. Since this is not realistic nor practical for an iGEM project, we decided on an alternative method. When supplemented with Cobinamide (Cbi, precursor for AdoCbl) E. coli is capable of producing on their own in small amounts. To achieve a greater yield of AdoCbl, the naturally occurring gene bluB of the synthesis pathway of sinorhizobium meliloti 2011 [3].

Proof of the production

To characterize the functionality of this part we first of all used Western Blot, an ethanolamine medium and mass spectrometry to qualitatively and quantitatively prove the production of AdoCbl in E. coli MG1655.

Western Blot to demonstrate expression relative to inducer strength

The Western Blot (decorated with an anti-His-Tag antibody, Fig. 2) confirmed the induced expression of BluB. Different concentrations of the inducer doxycycline were tested to identify the optimal yield of the BluB protein.

While we observed an increase in BluB protein yield with increasing inducer concentration, we also noted a leaky expression without induction. This is most likely due to a silent mutation that we introduced in TetR to conform to iGEM standards for this part.

Ethanolamine medium: An easy method to check for AdoCbl production

Ethanolamine medium is a minimal medium devoid of any nitrogen source despite ethanolamine. Since nitrogen is crucial for amino acid synthesis and ultimately cell survival, E. coli is unable to grow in such a medium. The use of ethanolamine as a carbon or nitrogen source requires AdoCbl, which is the cofactor of ethanolamine ammonia-lyase that catalyzes the conversion of ethanolamine to acetaldehyde and ammonia [4]. Cells can grow on ethanolamine only if there is AdoCbl available. Based on a publication from 1976 [5] we were able to produce a minimal medium in order to demonstrate the production of AdoCbl by overexpression of BluB. In this assay we compared the growth of piG_01b [BBa_K4604015] to the mutated non-functional bluB expression construct piG_07 [BBa_K4604020] after induced production in M9 medium.

Cells were cultivated in M9 medium, induced with 100 ng/ml DOX, and supplemented with the essential substrate for AdoCbl synthesis. After 24 hours, the cultures were washed and then cultivated in the ethanolamine medium to observe growth.

The growth curves clearly show that only the induced cells that contain the functional bluB gene (piG_01b) are capable of metabolizing the ethanolamine by producing AdoCbl. E. coli are able to produce AdoCbl from 3,3-Dimethyl-1-butanol (DMB) and Cobinamide (Cbi) alone, but only in small amounts since they do not produce enough BluB to synthesize large amounts of DMB. With added DMB or overexpression of BluB higher yields of AdoCbl can be obtained. This proves that bluB over-expression in E. coli leads to AdoCbl synthesis, which is crucial for ethanolamine metabolism.

Liquid Chromatography Mass Spectrometry (LC-MS) for quantitative testing

LC-MS is a method used to determine the concentration of molecules based on their mass. With this, it was possible to detect how much Hydroxocobalamin (OHCbl) is produced (relative to dry cell mass). When AdoCbl is exposed to light, it is converted to another derivative of B12, namely OHCbl. To make the preparation of the samples and measurement easier, we worked in sunlight, thereby converting AdoCbl into OHCbl, and measured the amount of this compound. We cultivated bacteria containing either a functional or a mutated version of the blub gene and afterward sent them to Hannibal Lab at the University Medical Center Freiburg, to be measured via mass spectrometry.

These results clearly show that the AdoCbl yield in our production culture is heavily increased by the overexpression of a functional BluB. With this method, we could prove the exact production quantities of AdoCbl our cells were able to synthesize. Reasonably, compared to highly calibrated systems [6], we produce less AdoCbl (530.29 μg/g dry cell weight vs. ~47.4 µg/g dry cell weight). Of course, higher yields can be achieved by further optimization of the conditions, however this was outside the scope of our research.

All these results state clearly that we were successfully able to achieve a production of AdoCbl in E. coli. See more detailed data on the B12 results page.

Riboswitch

Riboswitches occur naturally as regulators of gene expression. They are encoded as DNA sequences which exert their regulatory effect upon transcription by folding into a complex 3D RNA structure. Generally, a riboswitch can fold in two ways: Either with the ribosome binding site (RBS) being available or unavailable for the ribosome. Upon binding to the target compound, for example, AdoCbl, a configurational change in the riboswitch leads to the unavailability of the RBS. In E. coli, a riboswitch regulates the expression of the btuB gene (NIH: 948468) which encodes for a corrine transporter protein BtuB. It has been shown that sufficient AdoCbl concentrations cause the riboswitch to undergo a conformational change resulting in lower BtuB expression [7].

We decided on using this riboswitch to downregulate the toxin expression when AdoCbl is produced in sufficient amounts. To verify the performance of the riboswitch we created a sensor with a fluorescent readout inspired by the iGEM team Wageningen 2016 (BBa_K1913011). However, had we placed the sfGFP directly after the riboswitch, its negative regulation in the presence of AdoCbl would have resulted in a decrease in fluorescence. In our biosensor (BBa_K4604026) the riboswitch is placed in front of a repressor gene (lacI) which inturn suppresses sfGFP expression. If the riboswitch is triggered by binding AdoCbl, the repressor expression is stopped leading to a positive readout in the form of a detectable fluorescent signal.

The biosensor was tested independently of AdoCbl production. For this reason, we added different concentrations of AdoCbl to the cultures and tested the sensitivity of the sensor by fluorescent measurement. AdoCbl is taken up by the cells, binds to the riboswitch and enables the expression of sfGFP.

Fluorescent measurement, as shown in figure 7, indicates that at a concentration of 100 nM AdoCbl, the riboswitch is saturated and can no longer recognize an increase of AdoCbl. This data verifies the sensibility of the riboswitch; it can detect AdoCbl in different concentrations and accordingly regulate the gene downstream of it. However, we also noted a high background fluorescence without the addition of AdoCbl.

Toxin-Antitoxin System

Toxin-antitoxin systems (TA-systems) play a crucial role in plasmid stability for naturally occurring plasmids [8]. Usually, the toxin targets essential cellular functions and causes growth arrest or cell death, to which the antitoxin acts as a counterpart. Toxin and antitoxin exhibit differences in their stability and lifespan [9]. While the antitoxin has a shortened lifespan due to its sensitivity to degradation, the toxin has a longer lifespan and is more stable. If the plasmid containing the TA-system is lost, the antitoxin is rapidly degraded and the toxin concentration increases, leading to cell death. Therefore, when first discovered, TA systems were called “addiction modules” that ensure plasmid retention.

MazE/F as an executive force

The toxin-antitoxin system we chose is MazE/F. The labile MazE (antitoxin) acts as a neutralizer to the stable MazF (toxin), which is an endoribonuclease. When MazF is present freely in the cell it cuts cellular RNA which ultimately leads to cell death. It’s important to note that this system is already present and constantly expressed in the strain we used, therefore a baseline expression of the native system is given. The endogenous MazE/F system regulates its own gene expression [10]. We therefore decided to use a different promoter than the naturally occuring one to prevent interference by the toxin-antitoxin complex. To gain insights into the functionality of the antitoxin-toxin system we performed several experiments.

Proof of the inhibitory effect of MazF on cell growth

First, we needed to verify the functionality of the toxin MazF, for this purpose, it was tested separately from the AdoCbl production. In a separate construct, namely piG_23 (BBa_K4604024), we identified the time span in which the toxin MazF kills the cells and thereby confirmed its toxicity. This was accomplished by an assay consisting of OD measurements and colony-forming units (CFU). Cell toxicity was tested in a liquid culture with different inducer concentrations. First, a Western blot was performed to detect MazF expression.

Figure 8: MazF toxin expression for different inducer concentrations. Detection of recombinantly expressed, FLAG-tagged MazF toxin with SDS-PAGE followed by western blot using anti-FLAG antibody. Loading control: RNA polymerase subunit. Western blot with samples from different time points of pGGAselect in MG1655 in M9 medium, induced and uninduced in Lämmli buffer; Western blot with samples from different time points of piG_23 in MG1655 in M9 medium, induced in non-degrading buffer.

Induced expression of MazF was confirmed by western blot. The bands at 15 kD are the monomeric MazF. Since MazF also forms an unstable dimer, the faint bands at a height of 30 kD are presumably the MazF dimer. Moreover, we have observed an accumulation of MazF with increasing culture time. To determine the toxicity of the expressed MazF, we performed an assay on growth (fig. 9) and CFU (fig. 10)

Figure 9: Growth assay of E. coli MG1655, containing piG_23 induced with different DOX concentrations in M9 medium. OD600= 0.5 of culture samples were measured using ThermoScientific NanoDrop 2000c Spectrophotometer.	Figure 10: E. coli MG1655 colony forming units (CFU) assay. CFU/mL values for each timepoint for piG_23

A clear growth inhibition was observed in cultures of piG_23 (BBa_K4604024) induced with doxycycline concentration of 50 ng/ml and higher. To make sure that the toxicity is not due to the antibiotic nature of our inducer we tested different concentrations of doxycycline and their effect on the growth of cells not containing mazF.

Figure 11: E. coli MG1655 growth curve comparison in LB medium over 20 hours with different DOX concentrations.DOX added after an OD 600 of 0.4 was reached. OD 600 measurement of culture samples using ThermoScientific NanoDrop 2000c Spectrophotometer.

After this, we came to the conclusion that concentrations of up to 100 ng/mL do not inhibit cell growth. These results clearly prove the toxic effect MazF has on cells and their proliferation.

MazF regulated by the riboswitch

The next step was testing mazF in combination with the riboswitch to assess successful regulation of toxin expression. For this, we conducted a toxicity assay via OD measurements and CFUs. As opposed to the experiment shown above, this had to be done in M9 medium to prevent the riboswitch from being triggered by the AdoCbl concentration in LB-Medium. This caused a general decrease in growth.

Figure 12: Growth assay of E. coli MG1655, containing piG_03 over 24 hours in M9 medium with different DOX concentrations. OD600= 0.5 of culture samples were measured using ThermoScientific NanoDrop 2000c Spectrophotometer.	Figure 13: E. coli MG1655 colony forming units (CFU) assay. CFU/mL values for each timepoint for piG_03 and control pGGAselect.

These graphs indicate inhibited toxicity of MazF if it is regulated by the riboswitch. Our first assumption was that cells accumulated mutations in the toxin gene sequence due to the evolutionary pressure a toxin implies. However, after sending several samples of our cultures to sequencing, showing no mutations of the toxin, this thought was discarded. Consequently, we kept searching for a cause. The inhibited expression hinted at the ribosome binding site of the riboswitch being too weak for adequately high toxin production. This was further supported by the fact that the biosensor showed leakiness even in high concentrations of AdoCbl. This indicated that the repressor was being expressed in insufficient concentrations to inhibit GFP translation. Since we use different promoters in these plasmids we identified the RBS as the probable issue since it is the only regulatory element these plasmids share.

Decreased toxicity caused by RBS inherent in riboswitch

To investigate this hypothesis we modified the plasmid that proved to lead to cell death (piG_23a) by adding the ribosome binding site of the riboswitch in place of the present RBS (piG_23b/BBa_K4604025). A toxicity assay was performed again, following the same procedure as previously explained.

Figure 14A: Growth assay of E. coli MG1655, containing pGGAselect pGGAselect, piG_23 and piG_23b over 24 hours in M9 medium with different DOX concentrations. OD600= 0.5 measurement of culture samples using ThermoScientific NanoDrop 2000c Spectrophotometer.	Figure 14B: Growth assay of E. coli MG1655, containing pGGAselect pGGAselect, piG_23 and piG_23b(+Riboswitch RBS) over 24 hours in M9 medium with different DOX concentrations. OD600= 0.5 measurement of culture samples using ThermoScientific NanoDrop 2000c Spectrophotometer

The piG_23b containing cells, 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. This result indicates no difference in growth, while significant growth inhibition can be observed for piG_23 cells induced with 100 ng/ml DOX, as can be seen on piG_23 characterization. Constant OD600 values of around 0.8 indicate a stagnation in growth. Unexpectedly, we observed that piG_23 non-induced also showed signs of growth stagnation, so we replicated the experiment. Replicate 2 presented in Figure 14B showed a similar growth behavior as Replicate 1 (14A). We observed that the bacteria carrying piG_23b reached a higher OD although being induced with the same DOX concentration as the bacteria containing piG_23. In contrast to the last replicate, the non-induced piG_23 grew as expected, reaching similar ODs as the non-induced pGGAselect. However, for this repetition, we observed an unexpected growth of induced pGGAselect. This could, however, not be explained and it was the first time we observed this.

As shown in the graphs above the inhibitory effect of MazF is significantly decreased by adding the supposedly weaker RBS to the plasmids. This realization was a huge step forward in our research, yet finding a way to make MazF work with the riboswitch is a topic of further research.

Over the course of our project, we ran into several time limitations and could not successfully test all the parts of CELLECT. Since this was a predictable outcome, we parallelly tried a completely different approach to gain information, counteracting this lack of experimental data.

Modeling

Using parameters based on experimental results, observations and literature research, we were able to model this BioBrick in silico giving us a valuable insight into the underlying mechanics and dynamics of the system. To model CELLECT, we utilized conceptual models (CM). A conceptual model is a graphical representation of the components we want to track. Complex reaction networks can be well described by coupled Ordinary Differential Equations (ODE) since they can capture the dynamic behavior over time and the integration of quantitative data [11]. Our main findings using these methods were:

After combining all the variables in our system with a low constitutively expressed antitoxin we obtained the following plots:

As a result of the downregulation by AdoCbl binding to the riboswitch, one can observe the expression of toxin in less than lethal amounts. Further, one notices that for a final AdoCbl concentration of around 26 nM, the corresponding toxin concentration is quite high at around 65 µM. This suggests that higher AdoCbl concentrations in equilibrium lead to a decreased toxin expression, posing less of a metabolic burden to the cell. In order to determine the minimal AdoCbl concentration necessary for stress free conditions we look at the following plot.

Here we can observe how AdoCbl concentration in equilibrium affects toxin concentrations in equilibrium. Cell stress due to unbound and therefore enzymatically active toxin sets in at T>2A which is equivalent to a toxin concentration of 86.232 µM. An AdoCbl concentration in equilibrium of around 20 nM suggests that this is the minimal AdoCbl concentration necessary to prevent the generation of cell stress by toxin. The difference in AdoCbl concentration leading to either cell stress or cell death is negligible, suggesting that a minor change in AdoCbl levels has drastic effects. Important to note is that we see a decrease of toxin concentration with rising AdoCbl concentrations. This leads to an increased metabolic burden for non-producing E. coli, inturn giving high producing cells an evolutionary advantage over ones producing just the minimum concentration necessary required for survival. This could be seen as a form of directed evolution.

Conclusion/Future outlook

As shown by the given results, the parts work considerably well by themselves. We were able to test and verify their performance. What follows next is the most challenging part: composing the constructed parts to form a functional unit. Due to time constraints we did not manage to test the complete system. Nonetheless, there are still areas of improvement that pose a possibility for future research. One of which is the calibration of the delicate balance between the toxin and antitoxin concentration. Cells do an exceptional job at regulating the quantity of these molecules, which has to be mimicked for a practical autoregulatory system. Assumably, the promoters have to be adjusted or modified to reproduce the proportions of toxin to antitoxin in a stable manner. We did not implement the antitoxin in our experiments yet, since we assessed sole toxin experiments and a focus on other parts of the project more feasible and promising. Another part of the project that demands more attention, are the optimal conditions to further boost AdoCbl production. Ideal synthesis circumstances have to be identified to provoke an increased yield. This would also include testing how this BioBrick compares to established production methods after completed calibration. The problem every autoregulatory system eventually has to face is the fact that even after the desired production stops for whatever reason, the synthesized molecule is still present in the cells and thereby able to trigger the riboswitch. Since this BioBrick is regulated by recognizing the product, there is no way to distinguish molecules produced previously from newly synthesized ones. This can result in a lack of toxin expression even if the relevant reaction is no longer performed. Only after the compound is successfully broken down, consumed or secreted by the cells, the intended regulation can take place. Depending on the stability of the metabolite chosen in the final system, degradation times can vary.

References

[1] Casadesús J, Low DA. Epigenetic gene regulation in the bacterial world. Microbiology and Molecular Biology Reviews [Internet]. 2006 Sep 1;70(3):830–56. Available from: https://doi.org/10.1128/mmbr.00016-06

[2] Hallberg ZF, Su Y, Kitto RZ, Hammond MC. Engineering and In Vivo Applications of Riboswitches. Annual Review of Biochemistry [Internet]. 2017 Jun 20;86(1):515–39. Available from: https://doi.org/10.1146/annurev-biochem-060815-014628

[3] Fowler CC, Brown ED, Li Y. Using a Riboswitch Sensor to Examine Coenzyme B12 Metabolism and Transport in E. coli. Chemistry & Biology [Internet]. 2010 Jul 1;17(7):756–65. Available from: https://doi.org/10.1016/j.chembiol.2010.05.025

[4] Sheppard DE, Penrod JT, Bobik TA, Kofoid E, Roth JR. Evidence that a B 12 -Adenosyl Transferase Is Encoded within the Ethanolamine Operon of Salmonella enterica. Journal of Bacteriology [Internet]. 2004 Nov 15;186(22):7635–44. Available from: https://doi.org/10.1128/jb.186.22.7635-7644.2004

[5] Fa S, Jm T. Microbial Metabolism of Amino Alcohols. Ethanolamine Catabolism Mediated by Coenzyme B12-dependent Ethanolamine Ammonia-Lyase in Escherichia coli and Klebsiella aerogenes. Journal of General Microbiology [Internet]. 1976 Jul 1;95(1):173–6. Available from: https://doi.org/10.1099/00221287-95-1-173

[6] Dong L, Fang H, Gai Y, Zhao J, Jiang P, Lei W, et al. Metabolic engineering and optimization of the fermentation medium for vitamin B12 production in Escherichia coli. Bioprocess and Biosystems Engineering [Internet]. 2020 May 12;43(10):1735–45. Available from: https://doi.org/10.1007/s00449-020-02355-z

[7] Nou X, Kadner RJ. Adenosylcobalamin inhibits ribosome binding to btuB RNA. Proceedings of the National Academy of Sciences of the United States of America [Internet]. 2000 Jun 13;97(13):7190–5. Available from: https://doi.org/10.1073/pnas.130013897

[8] Ni S, Li B, Tang K, Yao J, Wood TK, Wang P, et al. Conjugative plasmid-encoded toxin–antitoxin system PrpT/PrpA directly controls plasmid copy number. Proceedings of the National Academy of Sciences of the United States of America [Internet]. 2021 Jan 22;118(4). Available from: https://doi.org/10.1073/pnas.2011577118

[9] Brzozowska I, Zielenkiewicz U. Regulation of toxin–antitoxin systems by proteolysis. Plasmid [Internet]. 2013 Jul 1;70(1):33–41. Available from: https://doi.org/10.1016/j.plasmid.2013.01.007

[10] Marianovsky I, Aizenman E, Engelberg‐Kulka H, Glaser G. The Regulation of the Escherichia coli mazEF Promoter Involves an Unusual Alternating Palindrome. Journal of Biological Chemistry [Internet]. 2001 Feb 1;276(8):5975–84. Available from: https://doi.org/10.1074/jbc.m008832200

[11] Motta S, Pappalardo F. Mathematical modeling of biological systems. Briefings in Bioinformatics [Internet]. 2012 Oct 14;14(4):411–22. Available from: https://doi.org/10.1093/bib/bbs061

Sequence and Features