Engineering & Results
- Part 1 : Inducible-Irreversible Genetic Switch
- Part 2 : Cross-kingdom Communcation
- Part 3 : [YLV2-CC]
- Part 4 : Biocontainment System
ENGINEERING & RESULTS
Part 1 : Inducible-irreversible genetic switch
DESIGN
[Figure1]
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The bioswitch we designed utilizes an inducible promoter and recombinase. The first design for this module, YLV2-AG, utilizes well-known strong galactose-inducible & glucose-repressible pGal1 promoters for the transcription of site-specific recombinase Cre from coliage P1. The genetic parts are located in such a way that the recombinase can recognize its recognition site loxPs and excise the sequences between them; the sequence coding for recombinase is excised with recombination so that the unneeded expression of recombinase can be restrained [Figure 1-A].
This way, YLV2-AG allows the target protein (GFP in this case) to be expressed only after the cell is introduced to the galactose (+) glucose (-) environment [Figure 1-B].
This way, YLV2-AG allows the target protein (GFP in this case) to be expressed only after the cell is introduced to the galactose (+) glucose (-) environment [Figure 1-B].
Build
The DNAs used in this research were obtained from iGEM kits, or synthesized by iGEM sponsors (Twist Bioscience Inc., San Francisco, CA, USA; Integrated DNA Technologies Inc., Coralville, IA, USA). Each promoter, coding sequences, and terminator is amplified by Q5 high-fidelity PCR (New England Biolabs Inc., Ipswich, MA, USA) using primers (Cosmogenetech co, Ltd., Seoul, Korea) with overhangs designed for golden gate assembly using BsaI-HFv2 (New England Biolabs Inc., Ipswich, MA, USA) and T4 ligase (New England Biolabs Inc., Ipswich, MA, USA). The yeast vector, with yeast/bacterial replication origin and KanMX antibiotic resistance, is amplified from pCEC-Red [1] purchased from Addgene (plasmid #196040). The assembled product is transformed to Stable Competent E. coli (New England Biolabs Inc., Ipswich, MA, USA) for amplification, and prepared using plasmid DNA purification kit mini (Labopass™ Cosmogenetech co, Ltd., Seoul, Korea). The yeast cells are transformed using LiAc/PEG/ssDNA method and assessed by yeast colony PCR. The fluorescence was measured by LUX multimode microplate reader (Varioskan™ Thermo Fisher Scientific Inc., Cleveland, OH, USA). The detailed protocol can be found in the experiment sub-section.
The amplification in E.coli through the transformation of the golden gate assembly product was not successful due to the recombinase activity in E. coli excising the sequences between the recognition sites. 50 bp homologous arms that match the yeast vector were designed and were mixed in the golden gate assembly mix instead of the vector. After very sufficient repetition of digestion and ligation, Q5 high-fidelity amplification using primers binding to each end of the homologous arm was conducted and verified through gel electrophoresis and sequencing of prepared DNA.
LEARN AND TEST
Switching promoter-terminator combinations to increase GFP expression.
[Figure 2]
The original design of YLV2-AG showed no significant difference in fluorescence expression after 96 hours of incubation in the galactose medium with G418 antibiotic selection. To inquire into the cause, the culture medium was washed in the glucose medium and streaked on the glucose agar medium. 5 colonies were randomly selected for colony PCR, and the result showed that all five colonies successfully underwent recombination of plasmid. In this regard, we found that increasing the expression of GFP could be a solution. The GFP expressions under constitutive promoter-terminator combinations were tested. According to the result, the ADH1 promoter - UBX6 terminator combination showed almost twice higher fluorescence expression compared to the ADH1 promoter - PRM9 terminator combination, which was in our original design. After switching to the UBX6 terminator, a significant difference in relative GFP expression in the galactose medium was observed after 96 hours of incubation [Figure 2].
Checking recombinase activity depending on incubation temperature & time
[Figure 3]
There has been research telling the activity of cre recombinase is maximized at temperatures higher than 37°C when tested in vitro [2]. The fluorescence was measured at 24h, 48, 72h, and 96h incubation at 30°C, 250 RPM and 37°C, 250 RPM for comparison. The result showed that the fluorescence expression of GFP is higher when cultured at 30°C than at 37°C [Figure 3]. The fluorescence data was normalized by dividing fluorescence (GFP 485/512) by absorbance (OD 600).
Part 2 : Cross-kingdom Communication
DESIGN
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The next bioswitch we are testing utilizes AHL, which enables bacteria to regulate gene expression depending on the population density through quorum sensing. Chimeric transcriptional activator is utilized for the expression of mutated Vibrio fischeri LuxR quorum sensing protein, and known LuxR-binding boxes are fused to pGal1-c minimal promoter [3] [Figure 4-A].
This way, yeast can recognize C6 AHL secreted from bacteria, thereby enabling gene expression only in the presence of the bacteria [Figure 4-B].
This way, yeast can recognize C6 AHL secreted from bacteria, thereby enabling gene expression only in the presence of the bacteria [Figure 4-B].
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In progress
Part 3 : [YLV2-cc]
DESIGN
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One problem with using YLV2-AG for our biocontainment system is that the expression of toxin begins during the initial incubation in the galactose (+) glucose (-) environment, which may have adverse consequences such as cell death or unintended selection. Adding purified AHL every time for the expression of antitoxin can be a solution, but is costly. One better solution can be using other combinations of promoters for module 1: pCTR3 and pCUP1, which are copper-repressible and copper-inducible promoters [Figure 5-A].
Utilizing their unique characteristic of being repressible and inducible for the same chemical, the expression of toxin after recombination can be controlled [Figure 5-B].
Utilizing their unique characteristic of being repressible and inducible for the same chemical, the expression of toxin after recombination can be controlled [Figure 5-B].
Learn and test
[Figure 6] Testing pCUP1 for YLV2-CC (BBa_K4406018)
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As a first step, each promoter was tested separately. The operability of pCUP1 in module 1 was tested by measuring the strength of GFP expression after induction by liquid YPD medium with G418 antibiotic and 0.1mM CuSO4 [Figure 6-A].
The GFP expression depending on recombinase activity in different culture periods (24h/48h/72h/96h), difference temperatures (30°C/37°C), and different preculture periods (0h, 8h, 24h) was measured as conducted with YLV2-AG, but no significant difference in GFP expression was observed in any conditions. To make an assessment at colony level, 24h culture at 30°C, 0h preculture, 250RPM was washed in liquid YPD medium and streaked on YPD agar medium with G418 antibiotic selection for 48h.
The colonies were assessed by colony PCR. Out of 10 colonies, 1 colony was found to underwent the recombination [Figure 6-B (right)]. The fluorescence of the colony and unsuccessful colony was assessed after 24h incubation at 30°C, 250RPM, showing a significant difference in GFP expression [Figure 6-B (left)]. The fluorescence data was normalized by dividing fluorescence (GFP 485/512) by absorbance (OD 600).
The GFP expression depending on recombinase activity in different culture periods (24h/48h/72h/96h), difference temperatures (30°C/37°C), and different preculture periods (0h, 8h, 24h) was measured as conducted with YLV2-AG, but no significant difference in GFP expression was observed in any conditions. To make an assessment at colony level, 24h culture at 30°C, 0h preculture, 250RPM was washed in liquid YPD medium and streaked on YPD agar medium with G418 antibiotic selection for 48h.
The colonies were assessed by colony PCR. Out of 10 colonies, 1 colony was found to underwent the recombination [Figure 6-B (right)]. The fluorescence of the colony and unsuccessful colony was assessed after 24h incubation at 30°C, 250RPM, showing a significant difference in GFP expression [Figure 6-B (left)]. The fluorescence data was normalized by dividing fluorescence (GFP 485/512) by absorbance (OD 600).
Learn and test
[Figure 7] Testing pCTR3 for YLV2-CC (BBa_K4406019)
The CTR3 promoter was synthesized in silico, using the known sequences of the upstream promoter region of the CTR3 [4]. The strength of GFP expression was measured [Figure 7].
[Figure 8]
The transformed colonies were cultured in two different liquid YPD medium with G418 selection, one with copper and the other one without copper, for 24h. The fluorescence microscopy results are as shown in the figure. In the presence of copper, the expression of GFP decreased significantly [Figure 8] .
Part 4 : Biocontainment system
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[Figure 9]
DESIGN
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[Figure 9]
We designed a biocontainment system using the two modules described above, replacing GFP with bacterial toxin RelE and antitoxin RelB from E. coli, known to work in S. cerevisiae [5]. The expression of RelE is controlled by module 1; the incubation in galactose (+) glucose (-) medium allows the recombination, enabling the expression of toxin [Figure 9-A].
The expression of RelB is controlled by module 2; the presence of AHL from bacteria allows the expression of antitoxin [Figure 9-B]. When yeast is separated from the bacteria, the expression of antitoxin ceases, making the yeast unable to survive [Figure 9-C].
The expression of RelB is controlled by module 2; the presence of AHL from bacteria allows the expression of antitoxin [Figure 9-B]. When yeast is separated from the bacteria, the expression of antitoxin ceases, making the yeast unable to survive [Figure 9-C].
- [1] Maestroni, L., Butti, P., Senatore, V. G., & Branduardi, P. (2023). pCEC-red: a new vector for easier and faster CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. Fems Yeast Research, 23. https://doi.org/10.1093/femsyr/foad002
- [2] Buchholz, F., Ringrose, L., Angrand, P., Rossi, F., & Stewart, A. F. (1996). Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination. Nucleic Acids Research, 24(21), 4256–4262. https://doi.org/10.1093/nar/24.21.4256
- [3] Tominaga, M., Nozaki, K., Umeno, D., Ishii, J., & Kondo, A. (2021). Robust and flexible platform for directed evolution of yeast genetic switches. Nature Communications, 12(1). https://doi.org/10.1038/s41467-021-22134-y
- [4] Peña, M. M. O., Puig, S., & Thiele, D. J. (2000). Characterization of the Saccharomyces cerevisiae High Affinity Copper Transporter Ctr3. Journal of Biological Chemistry, 275(43), 33244–33251. https://doi.org/10.1074/jbc.m005392200
- [5] Kristoffersen, P., Jensen, G. B., Gerdes, K., & Piškur, J. (2000). Bacterial Toxin-Antitoxin gene system as containment control in yeast cells. Applied and Environmental Microbiology, 66(12), 5524–5526. https://doi.org/10.1128/aem.66.12.5524-5526.2000