Team:NCKU Tainan/Design

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Design

In our mission to create a comprehensive solution to chronic kidney disease (CKD), we decided to focus on one of the roots of the problem, one that hasn’t been solved yet - the accumulation of p-Cresol in the human body.

In order to achieve our goals, we planned to engineer E. coli Nissle to have two specific functions. One, by diverting the original p-Cresol production pathway, we are able to convert tyrosine, the precursor of p-Cresol, into a beneficial substance for the body. Two, by producing a bacteriocin from a commonly used probiotic that can reduce the population of other Clostridium strains, which are the major p-Cresol producers. For biosafety reasons, we implemented an inducible kill switch by deleting the can gene and thus ensuring our E. coli Nissle can only survive in human gut.

During our research, we found out that Pseudomonas has the ability to sense p-Cresol. So we designed a p-Cresol (-GFP) reporter system by cloning Pseudomonas's p-Cresol sensing promoter to E. coli and culture it in our device for detection.

Fig. 1. The overview of Oh My Gut.

Alternative pathway

Tyrosine transporter (tyrP)[1]

tyrP encodes for tyrosine transporter, which transports tyrosine across the cytoplasmic membrane. We cloned it from E. coli MG1655 and designed a new biobrick which we then incorporated into E. coli Nissle, allowing it to take in more tyrosine. By doing so, our engineered E. coli can absorb excess tyrosine in the gut more efficiently, which in turn reduces the p-Cresol production by Clostridium.

Fig. 2. Plasmid design for uptaking tyrosine.

FNR promoter

The FNR (Fumarate and Nitrate reductase Regulatory) protein of E. coli is an oxygen-responsive transcriptional regulator required for the switch from aerobic to anaerobic metabolism. Under anoxic conditions, the FNR protein binds to DNA target sites and controls the expression of the corresponding genes[2].

In 2013, iGEM TU-Eindhoven used an artificial FNR promoter with two tandem FNR binding site, which has been reported to have a higher protein expression (1.8x) level under anaerobic condition[3]. We are concerned that TAL expression level may affect E. coli cell growth in normal conditions because it converts amino acids into other non-nutrient compounds. Also, due to the low oxygen level in the large intestines, where we want the protein to be expressed, we decided to use the FNR promoter in the TAL construct.

This is also the part that we are working on for part characterization. We repeated the experiment done by iGEM TU-Eindhoven 2013. However, after finding the characterization result was not consistent with our goal, we decided to abandon it.

How did we test it?

We measured the brightness of single E. coli cell under fluorescence microscope and use ImageJ Intensity Processing to quantify it. And compared the fluorescence intensity of bacteria in aerobic and anaerobic condition after 10 hours of incubation.

Tyrosine ammonia-lyase (sam8)

Tyrosine ammonia-lyase (TAL) is an enzyme that converts tyrosine into p-Coumaric acid. We engineered E. coli Nissle to express both TyrP and TAL to turn the excess tyrosine inside the gut into p-Coumaric acid[4], which is beneficial to humans. By reducing the total amount of tyrosine, the precursor of p-Cresol, we can reduce the amount of p-Cresol being produced by other gut flora.

Fig. 3. Plasmid design for converting tyrosine.

According to a research article that ranked different species of TAL[5], TAL from Saccharothrix espanaensis has been proven to have the highest conversion rate for tyrosine, and luckily, this enzyme has already been submitted as an iGEM biobrick Part:BBa_I742146. But, the iGEM biobrick contains a native ribosome binding site (RBS) from the original bacteria. We decided to change the native RBS into a strong RBS in iGEM collection B0034 to improve expression in E. coli Nissle.

Fig. 4. The mechanism of tyrosine transporter and tyrosine ammonia-lyase.

How did we test it?

In 2013, iGEM Uppsala has developed a method to measure TAL function[6]. By using NanoDrop UV-Vis spectrum and n-octanol extraction, we can extract p-Coumaric acid from the growth medium of bacteria and use its absorbance profile to quantify it. Therefore, we can test the tyrosine transporter with this same method. By growing TAL bacteria with and without the tyrosine transporter, we can then compare the results to determine how efficient our bacteria can be.

Reducing Clostridium population

Secretion tag (yebF)

YebF is a well-known E. coli secretory protein with unknown function. With a size of only 12 kDa, iGEM TAS Taipei 2015 and Seo EJ et al.,2012[7] have used it as a secretory tag for recombinant proteins. So, we decided to fuse it with our bacteriocin for secretion from our engineered E. coli Nissle.

Bacteriocin

Previous research has reported that Clostridium strains in the gut have the highest p-Cresol production rate, one of them being Clostridium difficile. So, we use it to demonstrate the p-Cresol production[8].

The research has also reported that a bacteriocin-like gene was found in a bacterium commonly used in probiotics and was proven to have bactericidal activity to certain strains of Clostridium. The bacteriocin (later called CBM-B) will self-cleave, and only the C-terminal region possesses bactericidal activity.

We designed three constructs, one full-length CBM-B fused with yebF, two constructs of C-terminal domain of CBM-B and yebF with two different linkers - a GS linker (three Glycine Serine repeat: GSGSGS) and a TB linker (thrombin cleavage site: LVPRGS).

Fig. 5. Plasmid design for reducing Clostridium population
Fig. 6. The mechanism of bacteriocin.

How did we test it?

We performed a spot-on-lawn assay using both growth medium and cell lysate of the bacteria carrying different bacteriocin constructs and observe the inhibition zone on the plate.

Biosafety

Knock-out of can gene[9]

The can gene of E. coli encodes a β-class carbonic anhydrase (CA), an enzyme that assists rapid interconversion of CO2 and water into carbonic acid, protons and bicarbonate ions. E. coli requires a constant supply of bicarbonate as a metabolic substrate during normal growth. So, if this gene is knocked out, E. coli is unable to turn the CO2 into bicarbonate fast enough before the CO2 diffuses out and cause cell death.

Thereby, we decided to use the can gene as a kill switch. Inside the gut, where we want the E. coli to survive, the CO2 concentration is high enough to allow the spontaneous conversion of CO2 into bicarbonate ions. However, when the E. coli exits the human body, the lowered concentration of CO2 will result in it’s death.

How did we test it?

We tested our knockout strain by placing them in different conditions, one in a normal incubator at 37℃ to simulate the normal CO2 concentration of the atmosphere, and one in a 5% CO2 incubator at 37℃ to simulate the CO2 concentration in the gut.

Knock-out of dapA gene[10,11]

DapA (4-hydroxy-tetrahydrodipicolinate synthase) is an enzyme necessary for cell wall synthesis. By deleting this gene, the bacteria will have to depend on exogenous diaminopimelate (DAP) for cell wall synthesis and growth, making it a kill switch as the bacteria will die without suppliance of DAP.

How did we test it?

To test whether E. coli can live with dapA condition and vice versa, we made LB plates containing DAP and knocked out dapA in E. coli Nissle with E. coli MG1655 as our control. We performed genotype check using colony PCR and phenotype check by comparing DAP plate and normal LB plate.

p-Cresol sensing system[12,14]

Initially, our project only consists of developing live therapeutics to provide an innovative solution to CKD. After consulting several doctors and other health care professionals, we realized that there was a need for the measurement of p-Cresol. Thus, we came up with CreSense - a p-Cresol biosensing device.

In a previous study[13], a particular promoter of Pseudomonas has the ability to sense p-Cresol. Our construct is primarily centered on Pseudomonas fluorescens (PC24), because it has a gene cluster that consists of a positive inducible operon, pchR, which allows it to sense p-Cresol and promote the transcription of the downstream region to degrade the p-Cresol. pchR encodes the activator proteins that can bind with the inducer (p-Cresol) and leads to the conformational change of the protein. As a result, the activator-p-Cresol complex will then bind onto the p-Cresol sensing region that is located downstream of pchR and start transcription. We replaced the downstream gene region with GFP, so it can sense p-Cresol and glow.

How would we test it?

We will grow the sensing bacteria on both LB agar plate and liquid culture with different concentrations of p-cresol and monitor the GFP emission intensity over time using a fluorescence plate reader.

Fig. 7. The mechanism of p-Cresol sensing system.

References

  1. Wolken, W. A. M., Lucas, P. M., Lonvaud-Funel, A., & Lolkema, J. S. (2006). The mechanism of the tyrosine transporter tyrP supports a proton motive tyrosine decarboxylation pathway in Lactobacillus brevis. Journal of Bacteriology, 188(6), 2198–2206. doi: 10.1128/jb.188.6.2198-2206.2006
  2. FNR regulon. (2019, March 1). Retrieved from https://en.wikipedia.org/wiki/FNR_regulon
  3. Barnard, A. M. L., Green, J., & Busby, S. J. W. (2003). Transcription regulation by tandem-bound FNR at Escherichia coli promoters. Journal of Bacteriology, 185(20), 5993–6004. doi: 10.1128/jb.185.20.5993-6004.2003
  4. Akdemir, F. E., Albayrak, M., Çalik, M., Bayir, Y., & Gülçin, I. (2017). The protective effects of p-Coumaric acid on acute liver and kidney damages induced by cisplatin. Biomedicines, 5(4), 18. doi: 10.3390/biomedicines502001
  5. Jendresen, Christian Bille, et al.(2015). Highly active and specific tyrosine ammonia-lyases from diverse origins enable enhanced production of aromatic compounds in bacteria and Saccharomyces cerevisiae. Applied and Environmental Microbiology, 81(13), 4458–4476. doi: 10.1128/aem.00405-15
  6. (n.d.). Retrieved from https://2013.igem.org/Team:Uppsala/p-coumaric-acid
  7. Seo, E.-J., Weibel, S., Wehkamp, J., & Oelschlaeger, T. A. (2012). Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins. International Journal of Medical Microbiology, 302(6), 276–287. doi: 10.1016/j.ijmm.2012.05.002
  8. Saito, Y., Sato, T., Nomoto, K., & Tsuji, H. (2018). Identification of phenol- and p-Cresol-producing intestinal bacteria by using media supplemented with tyrosine and its metabolites. FEMS Microbiology Ecology, 94(9). doi: 10.1093/femsec/fiy125
  9. Merlin, C., Masters, M., Mcateer, S., & Coulson, A. (2003). Why is carbonic anhydrase essential to Escherichia coli? Journal of Bacteriology, 185(21), 6415–6424. doi: 10.1128/jb.185.21.6415-6424.2003
  10. EMBL-EBI, I. P. (n.d.). InterPro. Retrieved from http://www.ebi.ac.uk/interpro/entry/IPR005263
  11. Rodionov, D. A., Vitreschak, A. G., Mironov, A. A., & Gelfand, M. S. (2003). Regulation of lysine biosynthesis and transport genes in bacteria: yet another RNA riboswitch? Nucleic Acids Research, 31(23), 6748–6757. doi: 10.1093/nar/gkg900
  12. Cho, A. R. (2011). Identification of a p-Cresol degradation pathway by a GFP-based transposon in Pseudomonas and its dominant expression in colonies. Journal of Microbiology and Biotechnology, 21(11), 1179–1183. doi: 10.4014/jmb.1104.04006
  13. Jõesaar, M., Heinaru, E., Viggor, S., Vedler, E., & Heinaru, A. (2010). Diversity of the transcriptional regulation of the pch gene cluster in two indigenous p-Cresol-degradative strains of Pseudomonas fluorescens. FEMS Microbiology Ecology, 72(3), 464–475. doi: 10.1111/j.1574-6941.2010.00858.x
  14. Jeong, J. J. (2003). 3- and 4-alkylphenol degradation pathway in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase. Microbiology, 149(11), 3265–3277. doi: 10.1099/mic.0.26628-0
  15. Flaticon, the largest database of free vector icons. (n.d.). Retrieved from https://www.flaticon.com/home.

e-mail : igem.ncku.tainan@gmail.com
phone : 06-2757575