Construct each part and test the function of TAL, tyrP, pchR and bacteriocin.
Using spot-on-lawn assay to test the bacteriocin bactericidal activity.
Use the previously developed method (see Protocols) to measure the p-Coumaric acid production of TAL.
Knockout dapA and can gene and phenotype check.
- Successfully proven CBM-B bacteriocin is able to inhibit Clostridium growth
In the human gut microbiome, Clostridium related species have been reported to have the highest conversion capability of fermenting tyrosine into p-Cresol. To target the root of p-Cresol accumulation, reducing the population of Clostridium is needed. We decide to use C. difficile as a model of p-Cresol producing bacteria because it is a popular research target due to its notorious infectious ability.
Luckily, iGEM NCKU Tainan 2019 was kindly supported by one of our PIs, Professor Huang, an assistant professor from the Microbiology and Immunology Department, who is currently devoting himself to the field of developing a novel therapeutic approach for C. difficile infection. He kindly provided us with a plasmid containing a bacteriocin gene (CBM-B), that was proven in his lab to have bactericidal activity against certain strains of Clostridium, including C. difficile.
As proof of the concept that bacteriocin is able to inhibit Clostridium growth, we did a spot-on-lawn assay using purified bacteriocin protein provided by advisors to observe the inhibition zone formation. As shown in Fig. 1 below, a clear inhibition zone formed in the middle of the BHI plate streak with C. difficile R20291 strain.
To further incorporate bacteriocin into our project, we wanted to integrate the bacteriocin into a live therapeutic drug instead of having patients consuming purified bacteriocins. Thus, we decided to fuse our CBM-B with a secretion tag, YebF. YebF is a secretory protein with unknown function in E. coli. We first amplified the yebF fragments from the genome of E. coli MG1655 and the CBM-B fragment from the plasmid kindly provided by Professor Huang. He confirmed that the CBM-B gene encodes a bacteriocin protein that will perform self-cleavage, and only the C-terminus fragment has bactericidal properties. So, we designed two versions of CBM-B construct by separately amplifying the full version of CBM-B gene and also the sCBM-B (short-length CBM-B) that encodes the C-terminus protein region only. Concerned that the fusion of sCBM-B with yebF may affect its bactericidal function, two linker sequences was introduced between yebF and sCBM-B using overhang primers. We also used PCR to amplify K880005 from iGEM distribution kit, and used overextension PCR to piece these fragments together, before ligating them into pSB3K3 plasmid.
However, after several attempts of constructing the yebF-bacteriocin plasmid, mutations kept appearing in the coding region. We will further complete yebF-bacteriocin construct in the future. We hypothesize that adding the yebF secretion tag into the construct is toxic to the bacteria.
Tyrosine ammonia-lyase & tyrosine transporter
- Construction of BBa_K2997000, BBa_K2997009 and BBa_K2997010
- Run SDS-PAGE to confirm expression in E. coli Nissle
- Conduct functional test to measure the activity of TAL and tyrP
We constructed tyrosine Ammonia-Lyase with a native RBS and B0034 RBS, (BBa_K2997009 and BBa_K2997010), and transformed both plasmids into E. coli Nissle 1917 and confirmed it by double digestion. The results are as follows:
Meanwhile, we amplified the tyrosine transporter and its promoter (BBa_K2997000) from E. coli MG1655 and cloning into pSB4A3. We then transformed the plasmid into DH5α and E. coli Nissle 1917.
RT-PCR experiment was performed to confirm the transcription of constructed TAL Biobrick. As shown in Fig. 8, cDNA for both bacteria carrying TAL constructs are being detected by PCR, confirming that the TAL gene is actually being transcribed in E. coli Nissle.
|1||PCR positive control||8||BBa_K2997009 cDNA||3+4|
|2||BBa_K2997009 cDNA||1+2||9||BBa_K2997010 cDNA||3+4|
|3||BBa_K2997009 RNA||1+2||10||BBa_K2997009 RNA||3+4|
|4||BBa_K2997009 plasmid||1+2||11||BBa_K2997010 RNA||3+4|
|5||BBa_K2997010 cDNA||1’+2||12||BBa_K2997009 plasmid||3+4|
|6||BBa_K2997010 RNA||1’+2||13||BBa_K2997010 plasmid||3+4|
Then, we carried out SDS-PAGE to check the protein expression of TAL, both with TyrP and without TyrP. The expected protein size of TAL is 54 kDa and the expected protein size of TyrP is 43 kDa. As seen in the results below, however, there’s no distinguishable band around both sizes.
Finally, to confirm the protein activity of TAL and TyrP, we performed a functional test using n-octanol extraction method, which was previously proposed by iGEM Uppsala 2013 and has been verified by HPLC. The p-Coumaric acid concentration was measured through the absorbance value at 310 nm wavelength under Nanodrop UV-Vis wavelength. The standard curve of p-Coumaric acid was drawn in Fig. 10 to determine the relationship between p-Coumaric acid concentrations and its 310 nm arbitrary unit (a.u.). Our samples with TAL constructs were then mapped onto the standard curve, to know how much p-Coumaric acid is being produced.
We compared the TAL constructs containing the native and B0034 ribosome binding sites, (BBa_K2997009 and BBa_K2997010) to determine if p-Coumaric Acid production is improved by changing the ribosome binding sites. From the results seen in Fig. 11, BBa_K2997010 is able to produce a higher amount of p-Coumaric acid. Hence, we are able to prove that by changing the RBS (from Native to B0034), the conversion of tyrosine into p-Coumaric acid can increase by 1.73-fold.
We can further improve the conversion of tyrosine into p-Coumaric acid by adding a tyrosine transporter (BBa_K2997000). As seen in Fig. 11, when tyrosine transporter is added, the production of p-Coumaric acid is significantly higher. When tyrosine transporter (BBa_K2997000) is introduced into E. coli Nissle with TAL constructs containing native RBS (BBa_K2997009) and B0034 RBS (BBa_K2997010), conversion of tyrosine into p-Coumaric acid is increased by 1.44-fold and 1.31-fold respectively.
To further prove that the TAL enzyme can specifically use tyrosine as its substrate, we cultured E. coli Nissle with dual plasmids containing TAL with B0034 RBS (BBa_K2997010) and tyrosine transporter (BBa_K2997009) in LB medium with different concentrations of tyrosine with hopes to see a dose-dependent effect. As seen in Fig. 12, although no significance in p-Coumaric acid production in culture supplemented with 0.5 mM and 1.0 mM tyrosine was detected, there was an increasing trend. Furthermore, there was a significant increase when comparing culture supplemented with 1.0 mM and 2.0 mM tyrosine. We speculate that there was indeed a dose-dependent effect, but due to the sensitivity limitations of n-octanol extraction method, it was not apparent.
- Construction of pchR-GFP BBa_K2997008 and confirm by double digestion.
The pchR-GFP (BBa_K2997008) was constructed from IDT DNA synthesis in the form of three gBlock gene fragments via overhang PCR. Then, we performed digestion on the finished part and ligated it into the pSB1C3 vector. Subsequently, we transformed the pchR-GFP construct (BBa_K2997008) into E. coli Top 10 using electroporation. After allowing the transformed E. coli Top 10 to grow on LB with Chloramphenicol plates, colony PCR and sequencing was carried out to determine successful clones. The colony PCR results indicated that the E. coli has pSB1C3 with pchR-GFP. However, after several attempts to construct pchR-GFP, all the sequencing resulted in mutations.
These results led us to hypothesize that the pchR protein is toxic to our E. coli. Moreover, according to a research paper we have found, it is suggested in the study that p-Cresol is toxic to Gram-negative bacteria, which includes E. coli. Hence, this plasmid construct has been dropped.
Therefore, the construction of pchR-GFP was carried out again by re-doing overlap PCR, where we combine all the three IDT gene fragments into the full-length pchR-GFP construct. For this time, the finished full-length pchR-GFP part was then inserted into the vector pBBR1 MCS-4 (contains ampicillin selection marker).
After that, we proceeded into transforming the construct into Pseudomonas fluorescens 55 and Pseudomonas aeruginosa PAD1 via electroporation.
However, due to several complications regarding the selection marker being used (Ampicillin), we were not able to obtain definitive results indicating that we have positive colonies. This is due to the fact that P. fluorescens 55 and P. aeruginosa PAD1 are naturally resistant to ampicillin and the vector we used contains ampicillin selection marker. Hence, positive colonies cannot be differentiated from negative colonies. Besides that, our colony PCR results on the single colonies were also proven to be negative.
Therefore, with time being one of the main factors, we decided to drop any further construction and functional experiments surrounding the p-Cresol sensing part (pchR-GFP).
- Successful knockout of can gene and dapA gene
In order to make our live therapeutic safe for human consumption, we added a kill switch into our E. coli Nissle in the form of gene knockouts. We used Lambda Red Recombineering system, which is based on homologous recombination, to perform gene knockouts. By using lambda red genes and hijacking E. coli Nissle’s own recombineering system, we are able to replace the target DNA region with a DNA fragment of our choosing. Electroporation was then used to deliver DNA fragments and plasmids into the bacteria.
We amplified the Chloramphenicol Resistance (CmR) Cassette from pKD3 plasmid using Amplification PCR, and used that fragment to replace the target gene. The resistance cassette is flanked by FRT sites, which then allows the removal of the cassettes with the help of pCP20, an FLP helper plasmid. Before we electroporated the CmR cassette into E. coli Nissle, we electroporated in pKD46, a recombinase helper plasmid.
We experimented with two different gene knockouts, can gene knockout and dapA gene knockout, and compared its effectiveness.
dapA Gene Knock Out
dapA encodes for 4-hydroxy-tetrahydrodipicolinate synthase, an enzyme necessary for cell wall synthesis. By knocking out this gene, the bacteria will have to depend on exogenous diaminopimelate (DAP) for cell wall assembly and growth, making it a potential kill switch as the bacteria will not survive without suppliance of exogenous DAP.
We confirmed that we have successfully replaced the dapA gene with CmR cassette using colony PCR. The size of CmR cassette inserted between the homology arms is approximately 1600 base pairs.
To further confirm our mutant E. coli works as a kill switch, we did a phenotype test by streaking out bacteria on different plates.
As seen in Fig. 16, without exogenous DAP, ΔdapA::CmR is unable to grow. And since it can survive on Cm plates, we can confirm that it contains the CmR cassette.
However, we realized that we would have to think of a way to provide exogenous DAP in our capsule to prevent our E. coli from dying before it reaches the gut. After deliberating the cost-performance ratio, we decided it wasn’t a good fit for what we had in mind. Thus, we did not continue on with replacing the CmR cassette with FRT sites.
can Gene Knock Out
The can gene of E. coli encodes for 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 causes cell death. Inside the gut, where we want the E. coli to survive, the CO2 concentration is high enough (ranging from 5% to 29%) to allow the spontaneous conversion of CO2 into bicarbonate ions. However, when E. coli exits the human body, the lowered concentration of CO2 will result in its death.
We confirmed that we have successfully replaced the can gene with CmR cassette using colony PCR. The size of CmR cassette inserted between the homology arms is approximately 1400 base pairs. The size of FRT sites inserted between the homology arms is approximately 500 base pairs.
To further confirm our mutant E. coli works as a kill switch, we did a phenotype test by streaking our bacteria on different plates and placing them in different conditions.
As shown in Fig. 18, Δcan::CmR and Δcan::FRT requires a higher CO2 level to survive. In doing so, we have proved that we have successfully knocked out the can gene.
Thereby, we will use the can gene knockout as our kill switch.
This year, we characterized FNR promoter (BBa_K1123000) which is an anaerobic promoter. Originally, we used this promoter to drive the expression of TAL constructs. However, finding that this promoter has a higher expression level of GFP under aerobic conditions, we did not use this promoter for our project. Instead, we used the strong constitutive promoter J23100 as our promoter.
We first cultured DH5α pSB1C3-Pfnr-GFP under aerobic and anaerobic conditions. After 10 hours of incubation, we fixed the E. coli cells in 2% agarose gel and placed it on a glass slide, then place the glass slide under a microscope for image capturing.
We then compared the brightness of a single E. coli cell under a fluorescence microscope using ImageJ Intensity Processing. The results are shown below.
As seen in Fig. 20, the fluorescence signal in single E. coli cell after 10 hours of incubation is significantly higher in aerobic conditions than in anaerobic conditions. Literature has reported that GFP requires oxygen molecules to fold properly before it can emit fluorescence signal. We cannot exclude the possibility that in anaerobic culture, GFP protein is not folding properly and thus affecting the measurement result. However, we can still conclude this promoter is not anaerobic specific. It is recommended that future iGEMers should use other reporter genes that will not be affected by oxygen like luciferase to further characterize this Biobrick.
- 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).
- Zhang, G., Brokx, S., & Weiner, J. H. (2005). Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nature Biotechnology, 24(1), 100–104.
- Passmore, I. J., Letertre, M., Preston, M. D., Bianconi, I., Harrison, M. A., Nasher, F., … Dawson, L. F. (2018). Para-cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria. PLoS pathogens, 14(9), e1007191.
- InterPro EMBL-EBI. “4-Hydroxy-Tetrahydrodipicolinate Synthase, DapA (IPR005263) < InterPro < EMBL-EBI.” Ebi.Ac.Uk, 2019, www.ebi.ac.uk/interpro/entry/IPR005263. Accessed 5 July 2019.
- Merlin, C., Masters, M., McAteer, S., & Coulson, A. (2003). Why Is Carbonic Anhydrase Essential to Escherichia coli? Journal of Bacteriology, 185(21), 6415–6424.
- Hashimoto, M., & Kato, J.-I. (2003). Indispensability of the Escherichia coli Carbonic Anhydrases YadF and CynT in Cell Proliferation at a Low CO2 Partial Pressure. Bioscience, Biotechnology, and Biochemistry, 67(4), 919–922.
- Coralli, C., Maja Cemazar, Chryso Kanthou, Tozer, G. M., & Dachs, G. U. (2001). Limitations of the Reporter Green Fluorescent Protein under Simulated Tumor Conditions. Cancer Research, 61(12), 4784–4790.