Team:Thessaly/Results
Results
Overview
Because of the unique situation this year, our access in the lab was limited. However, we started building our designed modules to lay the groundwork for future experiments. To accomplish that, we repurposed the GoldenBraid modular assembly strategy to enable bacterial expression of multigenic constructs, utilizing the SEVA standardized plasmid collection. We then assembled the GPCR-Tango Module, the Tet-off Module, the Prom Module and the Tyrosine-Tyrosinase Reporter Module that comprise the Monitoring System of Amalthea. Finally, we characterized and tested both Prom Module and Tyrosine-Tyrosinase Module, to prove that our system works.
Strategy
Our experiments were designed in a way that we could build new genetic Modules combining basic DNA parts in a standardized way. In order to increase the multigene assembly efficiency and perform successfully our experiments following the GoldenBraid assembly strategy that is used for Plant Synthetic Biology. This strategy is based on Type IIS restriction enzymes allowing the creation of our genetic circuits or our Modules. We modified our basic DNA parts designing compatible sites, in order to combine them and create final constructs that consist of a promoter, a CDS, and a terminator and our final Modules with this repeated pattern. In that way, we built an increasingly complex multigene System that can facilitate the heterologous expression of proteins in bacteria.
Level 0 cloning
The iGEM parts are the legacy of every Team for the future iGEM Teams. Using the iGEM registry is the best way to build your experimental design. This year we submitted several interchangeable Level 0 parts based on the TypeIIS Assembly. We include both project-specific and general parts, which are compatible for GoldenBraid and MoClo. This way, we make a contribution for future iGEM Teams.
Promoters
Figure1. (U=Uncut , C= Cut) Restriction digestion of AndersonJ23115 with : EcoRI (C1a & C2a),Expected bands : 2156 bp, XhoI (C1b & C2b),Expected bands : 1264 + 892 bp Positive result: The set of C1a and C1b (same sample).
Figure 2. (U=Uncut , C= Cut) Restriction digestion of TetO (C1-C4 ) with : Not-HF(C1-C4) , Expected bands : 2046 + 129 bp ,Positive result: C1,C2,C4
Figure 3. (U=Uncut , C= Cut)Restriction digestion of LacO (C3 & C4) with : EcoRV + HindIII (C3 & C4) , Expected bands : 2173bp ,Positive result : C3 + C4
Figure 4. Restriction digestion of pFlic (C1-C6) with : with SlaI, Expected bands: 1314 bp + 892 bp, and EcoRI, Expected bands: 2206 bp. Positive result : C5 + C6.
Figure 5. (U=Uncut , C= Cut) Restriction digestion of PrpB (C1 & C2) with : EcoRV + HindIII (C1 & C2) , Expected bands : 2297bp ,Positive result: C1 & C2
Figure 6. (U=Uncut C=Cut) Restriction Digestion of araC:ParaBAD with EcoRI+ PstI ,Expected bands: 2029+1996 , Positive result
Coding secuences
Figure 7. (U=Uncut C=Cut) Restriction Digestion of LacI with EcoRI + SlaI, Expected bands : 3189 +2300 + 892 bp, Positive Result:C2
Figure 8. (U=Uncut C=Cut) Restriction digestion of TetR (C2 -C4) with : SpeI + EcoRI (C2 - C4) , Expected bands : 2047 + 680 bp Positive result : C4
Figure 9. (U=Uncut C=Cut) Restriction digestion of RraA (C2 ) with : PstI + EcoRI , Expected bands: 2029, 564 bp
Restriction digestion of DjlA (C4 )with EcoRI, PstI Expected bands 2029, 894 bp
Figure 10. (U=Uncut C=Cut) Restriction digestion of sfGFP (C4 ) with : EcoRI, PstI Expected bands 2029, 794
Figure 11. (U=Uncut C=Cut) Restriction digestion of ECFP (C1-C3 ) with : SlaI, Expected bands : 1934 +892 bp, Positive Result : C1,C3
Figure 12. (U=Uncut C=Cut) Restriction digestion of EGFP (C1-C4 ) with : PvuII, Expected bands : 2826 bp , Positive Result : C4
Figure 13. (U=Uncut C=Cut) Restriction digestion of Tyr-Aida (C1-C4 ) with : EcoRV, EcoRI, BamHI. Expected bands 1378, 896, 700, 550, 490, 430, 200. Positive result : C
Figure 14. (U=Uncut C=Cut) Restriction Digestion of b-arrestin-TEV protease with SlaI, Expected bands : 2014 bp + 892 bp + 760 bp + 394 bp, Positive Result: green arrows
Figure 15. (U=Uncut C=Cut) Restriction Digestion of FFAR2:V2tail:TCS with BamHI, Expected bands : 3204bp, Positive Result:C2,C3
Figure 16. (U=Uncut C=Cut) Restriction digestion of Terminator (C3 )with : EcoRI & PstI ( C3 ), Expected bands : 2029 + 209 bp , Positive result : C3
Vectors
To facilitate standardized and modular cloning in our experiments. We built four standardized vectors, which are a bacterial-expression variation of the GoldenBraid standard (Sarrion-Perdigones et al. 2013), combined with SEVA plasmids. Using our system, the whole SEVA collection can be utilized to create vectors with a desirable of antibiotic resistance genes and ORIs by targeting the T0 and T1 terminators that are universal. Explore our full design here
Assembly of bacterial GoldenBraid vectors
pSEVAb23 (Damalas et al., 2020) was used as a backbone for the pGBD1 alpha 1R and pGBD1 alpha 2 using Q5 (NEB) PCR amplification because Level cloning has kanamycin resistance. Resulting in a backbone with kanamycin and BsmBI BsaI sites. The backbones were amplified with the primers from Table 1. Then Agarose Gel Electrophoresis (AGE) is conducted. And after that Gel extraction and Clean up isolating the backbones with the overhangs.
Then with BsmBI restriction digestion, the LacZa module was taken from the pDGB1 alpha vectors and with BsaI from the pDGB1 omega vectors. The whole reactions were loaded and AGE was conducted. Then Gel extraction and Clean up isolating the LacZa insert.
The same for pSEVAb43 that was used as the backbone for the pGBD1 omega 1R and pGBD1 omega 2 using PCR amplification because Level cloning has spectinomycin resistance. Resulting in a backbone with spectinomycin and BsmBI BsaI sites.
Digestion-Ligation with BsmBI and T4 Ligase (NEB for GB cloning) Backbones and Inserts were combined resulting in our E. coli friendly Plasmids (Figures 17, 18).
| CPrimer | Sequence from 5’ to 3’ |
| Alpha 1R FORWARD | ATctcctgagacggatccagtaggacaaatccgccgcc |
| Alpha 1R REVERSE | ATgtcatgagacggatccttgttcagaacgctcggttgcc |
| Alpha 2 FORWARD | ATtgactgagacgaagcttagtaggacaaatccgccgcc |
| Alpha 2 REVERSE | ATcgcttgagacgaagcttttgttcagaacgctcggttgcc |
| Omega 2 FORWARD | ATtgactgagaccgatatcagtaggacaaatccgccgcc |
| Omega 2 REVERSE | ATcgcttgagaccgatatcttgttcagaacgctcggttgcc |
| Omega 1 FORWARD | Tgcggccgcggatatattgtggtgtaacg |
| Omega 1 REVERSE | Tgcggccgcccaatatatcctgtcaggat |
Figure 17. A) pDGB1 alpha 1R vector with SEVA23 backbone B) pDGB1 alpha 2 vector with SEVA23 backbone.
Figure 18. A) pDGB1 omega 2 vector with SEVA43 backbone B) pDGB1 omega 1R vector with SEVA43 backbone.
Verification of our constructs
As it is showed at Figure 17 and 18B the 3 vectors pDGB1 alpha 1R, pDGB1 alpha 2, pDGB1 omega 2 were correct.
Figure 19. pDGB1 a1R digested with BamHI.
Expected bands: 2847 bp + 385 bp + 239 bp.
pDGB1
a2 digested with HindIII. Expected bands: 2847 bp + 416 bp + 206 bp.
Figure 20. A) pDGB1 omega1R digested with BamHI. Expected bands: 3153 bp + 382 bp + 239 bp. B) pDGB1 omega2 digested with EcoRV and HindIII. Expected bands: 2900 bp + 398 bp + 223 bp.
But the vector pDGB1 omega 1R had a problem on its primers’ set so the reverse idea was conducted. We designed a new set of primers having overhangs with NotI (Table 1). We amplified the LacZa module including the BsmBI BsaI sites from the original pDGB1 omega 1 vector. Then we digested with NotI the pSEVA43, in order to take the backbone.
At the beginning we had designed primers for pDGB1 omega 1, but cutting with NotI the LacZa module with the BsmBI and BsaI sites could be ligated in either orientation. Through sequencing we confirmed that it was ligated with reverse orientation (Figure 20 A). So omega 1R is our final vector. This result has no problem with the cloning though. In the future, we will replace our current ORI (pBBR1), which provides medium/low copy numbers, with a high copy one to facilitate cloning of complex constructs. This is easy, because according to the SEVA standard, we will digest with rare restriction enzymes PshAI and SwaI.
Tango-GPCR Module
The potential of SCFAs as diagnostic and therapeutic biomarkers can be utilized, by engineering a GPCR-based artificial signaling pathways in E. coli.This synthetic signaling pathway is based on the TANGO high-throughput screening technology, that takes advantage of the b-arrestin capabilities to bind to the GPCR receptor. For the whole design click here.
Assembly of Tango-GPCR Module
The Tango module consits of 4 TUs. The first is an arabinose inducible synthetic FFAR linked with LacI as seen at (Figure 21) based upon the work done by (Barnea et al. 2008) The second TU is a fusion of the b-arrestin-2 with the TEV protease, which liberates the LacI from the GPCR bioreceptor as seen at (Figure 22) The third TU is a Lac regulated Expression of eCFP see(Figure 23) The fourth TU is an arabinose inducible antitoxic proteins seen at (Figure 24) We have verified the correct assembly both by diagnostic digestions and Sanger Sequencing. Our goal is to test this system for iGEM 2021.
Verification of Tango-GPCR Module
Figure 21. (U=Uncut , C= Cut) Restriction digestion a1R:ParaBAD:RBS-FFAR2:V2tail:TCS-Lac-Double terminator (C1a-C4b) with : BamHI(C1a-C4a) , Expected bands : 2847+2225 bp , EcoRV (C2a-C2b) ,Expected bands : 3587 bp + 2845 bp, Positive result: C1,C2,C3,C3 (C1a and C1b is the same sample etc)
Figure 22. (U=Uncut , C= Cut) Restriction digestion of ParaBAD:RBS:B-arrestin -TEV protease -Double Terminator (C1-C 4), with :HindIII (C1a-C4a), Expected bands : 2847+2490+719 bp , EcoRV (C1b-C4b) , Expected bands:3988 bp+2214 bp. Positive result: C1,C2,C3,C4. (C1a and C1b -same sample etc)
Figure 23. (U=Uncut , C= Cut) Restriction digestion of AndersonJ23115:LacO -ECFP -Double Terminator (C1-C 4), with : BamHI (C1-C4) , Expected bands:2847 +952 bp. Positive result: C1,C2,C3,C4.
Figure 24. Restriction Digestion of ParaBAD-DjlA-Terminator (C1-C2) with EcorV and BamHI ,Expected bands 4212+ 853 bp, Positive Result : C1 Restriction Digestion of ParaBAD-RraA-Terminator (C3-C4) with EcoRV Expected bands 3471+1264 bp , Positive Result C4
Tet-off Module
Our aim was to reduce the complexity of bacterial GPCR expression and we designed a simple NOT-GATE system induced by anhydrotetracycline (aTC). Behind this module there is the same logical structure with the GPCR -Tango Assay. Α modular system that can simulate the detection of absence of SCFAs from the GPCR. Tetracycline Repressor (TetR) is a well-characterized transcriptional factor and we can make use of a tetracycline derivate, anhydrotetracycline (aTC) a strong chemical repressor (Gossen and Bujard, 1993). For full design click here.
Assembly of Tet-off Module
This module consists of several individual Level α transcriptional units (TUs). In order to create the NOT-GATE device, we assembled a constitutive expression TetR TU as seen at (Figure 26), a LacI-regulated ECFP TU AndersonJ23115:lacO-eCFP-double terminator (Figure 23) , and a tet-regulated expression of Lac inhibitor.
Verification of Tet-off Module
Figure 25. (U=Uncut , C= Cut) Restriction Digestion of TE: AndersonJ23115:TetO-EGFP-double terminator with EcoRV(C1-C2). Expected bands : 2847+ 954 bp. Positive result: C1,C2.
Figure 26. (U=Uncut , C= Cut) Restriction Digestion of T: AndersonJ23115-TetR-double terminator digested with HindIII (C3-C4). Expected bands : 2847 + 836 bp. Positive result : C3,C4.
Figure 27. (U=Uncut C=Cut) Restriction digestion of TL: AndersonJ23115:Tet0-LacI-double terminator(C5-C8 ) with: HindIII + NheI (C5-C18) , Expected bands : 2573+891+427,Positive result : C5,C6
After level α constructs were completed , and more specifically the AndersonJ23115:LacO-eGFP-double terminator (Figure 23) and AndersonJ23115:TetO-eGFP-double terminator (Figure 24) , we started plate-reader assays before sequencing results arrived. This construct is crucial for our experiments as it used to the majority of our modules. After sevreal measurements that yielded no fluorescence, we came to a conclusion that something was wrong with this transcription unit as no detectable fluorescence was produced in our initial experiments testing the eGFP. After the sequencing result came in , we noticed single point mutations in our eGFP clones. After we BLASTed the sequences, eCFP was 100% identical with the sequencing sample.It turns out that due to (most likely) wrong starting template, we had cloned eCFP instead of eGFP, which was the reason for non-detectable fluorescence. The next step was to adapt our protocol to new standards and create a new protocol.
Measurements are the average of 9 total replicates (3 biological replicates and 3 technical replicates per biological replicate). Error bars represent standard deviation of biological replicates.
The results of the assay can be seen in the figures below:
Figure 28. Validation that J23115-TetO and J23115-LacO fusion promoters are able to drive expression of ECFP.Expression of ECFP, after 16h of incubation at 37oC , using M9 Medium.
Figure 29. Bacterial Growth after 16h incubation at 37 oC with M9 medium.
Prom Module
We designed a a promoter-based module for SCFAs detection that performs as a NOT-GATE devices, in case the GCPR-Tango module is not functional. We used SCFA-induced promoters, pFliC (Tobe, Nakanishi and Sugimoto, 2010) and prpBCDE (Tsang, Horswill and Escalante-Semerena, 1998), so as two SCFAs work as an input. Downstream of them is a transcriptional factor, LacI, which binds to a Lac Operator, when is activated, leading to the inhibition of the expression of the reporter gene. Explore our full design here
Assembly of Prom Module
Our goal was to create our final constructs that will consist of our promoter, LacI, LacO and the reporter gene, in order to create a NOT-GATE. To do so we created different constructs for the expression of LacI and the expression of our reporter gene, eCFP. The expression of LacI was regulated by our SCFAs-induced promoters as you can see at (Figures 30, 31), while our reporter gene, eCFP was downstream of Lac Operator (LacO)(Figure 23).These constructs were combined into a final omega1R as seen at Figure 36, creating a NOT-GATE and allowing us to test our Module. For more information about our Proof of Concept here
Verification of Prom Module
Figure 30. Restriction Digestion of LEVEL 1: prpB-lacI-terminator with HpaI (C1-C2). Expected band: 4286 bp. Positive result : C1 .
Figure 31. Restriction Digestion of LEVEL 1: pFliC-LacI-terminator digested with BamHI (C1), Expected bands: 2847 bp + 1348 bp, and with HpaI (C4) , Expected bands 4195 bp. Positive result : C1 +C4
We created Level A constructs that consisted of our promoter, pFlic or prpBCDE, LacI and terminator into an alpha1R vector and a constitutive promoter of iGEM Registry, Anderson J23115, combined with the Lac Operator (LacO), eCFP and terminator into alpha2 vector.(Figure 23)
Figure 32. Restriction Digestion of omega 1R pFliC:RBS-LacI-Terminator-pAndersonJ23115:LacO:RBS-ECFP-terminator with EcoRV+ HindIII (C1-C3). Expected bands: 3734 + 1699 bp. Positive result : C2.
Then Level A constructs were combined together, into omega1R vector creating our final constructs and our Prom Module regarding pFliC promoter that we further characterized for iGEM. For more information about our Characterization here
Then we transformed this construct into MC1061 bacteria strains, as they are our go-to stains for protein expression of our reporter gene. Our goal was accomplished and then we had to test the function of our NOT-GATE. We added SCFAs based on the gathered data from our Characterization Read more, modeling Read more and observed the decrease of the expression of eCFP at different time-points see (Figures 33-36).
Figure 33. NOT-GATE-regulated ECFP fluorescence after in the presence or absence of 2mM acetate.
Figure 34. Cell growth in the presence or absence of 2mM acetate.
Figure 35. NOT-GATE-regulated ECFP fluorescence after in the presence or absence of 2mM propionate.
Figure 36. Cell growth in the presence or absence of 2mM propionate.
The results show that there is a noticeable difference regarding the expression of eCFP, when we add 2mM acetate or propionate. We hypothesize that the gap will be bigger when adding butyrate, which is the main inducer for pFliC, and which we didn’t have in the lab as per Wiki Freeze date. However, our data indicate that changing the conditions, adding acids, affect our Module in the way we expected as it acts as a NOT-GATE circuit.
For more information click here
Reporter Module
Tyrosine-Tyrosinase is a reporter module that has the ability of converting the presence of L-Tyrosine to an electrochemical signal for convenient readout. For extensive experimental design click here.
For extensive experimental design click here.
Assembly of Reporter Module
Tyrosinase has the ability of converting L-Tyrosine to L-Dopa and L-Dopa quinone which is electrochemically detectable (Figure37). VanArsdale expressed and measured a Tyrosine fused with an autotransporter for membrane transportation under control of an inducible promoter(VanArsdale et al., 2020).
We constructed a constitutive expression of Tyrosinase as seen at Figure 41, expecting Tyrosinase to stay on the membrane and wait for L-Tyrosine to appear.
Verification of Reporter Module
Figure 37. Restriction Digestion of pAndersonJ23115:RBS-tyrosinase:AIDA-terminator with BamHI(C1-C3). Expected bands 2847+1883+520+353 bp. Positive result : all.
We transformed this construct into MC1061 expression bacteria and conducted a Tyrosinase assay as seen in (Figure 38). We added exogenous L-Tyrosine after the expression of our protein, while included appropriate controls to obtain a specific signal. The measurements resulted in no detectable output, as the same measurements were captured either from Bacteria that had the Tyrosinase or no proper Insert, as seen at (Figure 38).
We hypothesize that constitutive expression of the proteins leads to toxicity and subsequent impaired cell growth, as seen in (Figure 39). Notably, several biological replicates of our insert grew at half the speed compared an empty vector control.In our next Design-Built-Test cycle, we will include an Arabinose inducible promoter to replace the constitutive one. The choice of the specific promoters has two main reasons: 1) We already have a verified clone in the lab, so using out modular assembly system we can just replace the promoters, and 2) Our goal is to overexpress Tyrosinase along with GPCR expression in our GPCR-Tango Module, to synchronize SCFA detection and signal production. Inducing by the same promoter can provide this feature.
Figure 38. Testing the activity of Tyrosinase measuring the formation of melanin after the conversion of L-Tyrosine to L-dopa.
Figure 39. Bacterial Growth after 6h at 30oC incubation with Tyrosinase Assay Buffer.
References
Alejandro Sarrion-Perdigones, Marta Vazquez-Vilar, Jorge Palací, Bas Castelijns, Javier Forment, Peio Ziarsolo, José Blanca, Antonio Granell, Diego Orzaez (2013). “GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology.” Plant Physiology , 162 (3) 1618-1631; DOI: 10.1104/pp.113.217661
Barnea, G., Strapps, W., Herrada, G., Berman, Y., Ong, J., Kloss, B., Axel, R., & Lee, K. J. (2008). The genetic design of signaling cascades to record receptor activation. Proceedings of the National Academy of Sciences of the United States of America, 105(1), 64–69. https://doi.org/10.1073/pnas.0710487105
Damalas, S., Batianis, C., Martin‐Pascual, M., Lorenzo, V. and Martins dos Santos, V., 2020. SEVA 3.1: enabling interoperability of DNA assembly among the SEVA, BioBricks and Type IIS restriction enzyme standards. Microbial Biotechnology, 13(6), pp.1793-1806.
Gossen, M. and Bujard, H., 1993. Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells. Nucleic Acids Research, 21(18), pp.4411-4412.
Tobe, T., Nakanishi, N. and Sugimoto, N., 2010. Activation of Motility by Sensing Short-Chain Fatty Acids via Two Steps in a Flagellar Gene Regulatory Cascade in Enterohemorrhagic Escherichia coli. Infection and Immunity, 79(3), pp.1016-1024.
Tsang, A., Horswill, A. and Escalante-Semerena, J., 1998. Studies of Regulation of Expression of the Propionate (prpBCDE) Operon Provide Insights into How Salmonella typhimurium LT2 Integrates Its 1,2-Propanediol and Propionate Catabolic Pathways. Journal of Bacteriology, 180(24), pp.6511-6518.
VanArsdale, E., Hörnström, D., Sjöberg, G., Järbur, I., Pitzer, J., Payne, G., van Maris, A. and Bentley, W., 2020. A Coculture Based Tyrosine-Tyrosinase Electrochemical Gene Circuit for Connecting Cellular Communication with Electronic Networks. ACS Synthetic Biology, 9(5), pp.1117-1128.
