Oxygen Sensing
Overview
Goal
To construct an oxygen sensing system using oxygen-responsive promoters pAnb1 and pRox1 from Saccharomyces cerevisiae that can be applied to wound monitoring.
Result
Differences between aerobic and anaerobic conditions were indicated by fluorescence protein using S. cerevisiae with the constructed oxygen detection system. However, oxygen-dependent change of fluorescence was also observed in the constitutive promoter(pCyc:BBa_I766555).
Future Direction
It is necessary to re-examine whether the change of fluorescence is due to promoters or a decrease in metabolic activity in the cells by setting up proper controls, like ADH1 promoter. And also, the threshold of the sensor system has to be quantified by using an oxygen concentration meter. Furthermore, creating a system that can be applied to coating materials is expected for practical use. This can be possible by incorporating chromoproteins or other reporter genes visible to the naked eye downstream of the oxygen promoter.
System Design
We aimed to construct a sensor system by hacking the oxygen response pathway in S. cerevisiae, and the first step was to determine which promoters to use. Upstream regions of ANB1 and ROX1 are promoters which have responsiveness to changes in oxygen concentration, and their expression is activated under aerobic conditions in plasmid conditions [1][2][3]. In this system(see the Description), pAnb1 and pRox1 were used, and fluorescent protein mCherry is attached downstream of pAnb1 and pRox1, and the CYC1 terminator was placed downstream of mCherry. This system reports the change of oxygen concentration by expressing fluorescent protein.
The information of each construct is shown inTable1. A sequence in pROX1 was modified to conform to BioBrick specifications.
Table 1 Promoter information
| Name | Length | Sequences |
|---|---|---|
| Oxy sensor pAnb1 | 2286bp | ANB1 promoter - mCherry - CYC1terminator |
| Oxy sensor pRox1 | 1565bp | ROX1 promoter - mCherry - CYC1terminator |
Parts Construction
The oxygen sensing system plasmid was constructed based on the design above. First, a plasmid in which the CYC1 promoter-mCherry-CYC1 terminator was incorporated into the MCS of the BBa_K1680016 backbone a yeast-E. coli shuttle vector was prepared. The following linear backbone DNA was extracted from the plasmid by PCR. The target plasmid was obtained by combining the sequences of pROX1 (synthesized by IDT) and pANB1 (cloned from S. cerevisiae BY4741 strain) with the linear backbone by the SLiCE method, respectively. By transferring this plasmid into S. cerevisiae BY4741 strain, we constructed the yeast strains which have the oxygen sensing system.
Oxygen Assay
Using the yeast strains established above, we observed the change of fluorescence under aerobic and anaerobic conditions.The pAnb1, pRox1, and mCherry strains (constitutively expressing mCherry) were cultured under anaerobic and aerobic conditions for one day each, and observed by a microscope (40x NA0.95).
Table 2 Experimental setup
| Condition | Experimental setup |
|---|---|
| Aerobic condition | Incubate overnight with shaking in a normal tube for yeast incubation. |
| Anaerobic condition | Incubate overnight with shaking in a 5ml syringe filled with medium. |
Fig. 1 Experimental setup of anaerobic (left) and aerobic condition(right).
Below(Fig. 2) is a microscopic image obtained from the above experiment. As can be seen from this image, the expression of mCherry is repressed in both pROX1 and pANB1 strains under anaerobic conditions.
Fig. 2 Change of fluorescence by pAnb1, pRox1, and Constitutive(pCyc) promoter under aerobic and anaerobic conditions. (40x NA0.95, BF:50ms, Fluorescence:600ms)
In addition, segmentation of cells was (segmented by cellpose [4] from Phase image) performed on the captured images to quantify the distribution of fluorescence states for each cell (Fig. 3). Intense fluorescence (over 1000) was observed only in aerobic conditions in any strains but in control and pRox1 strains change of fluorescence intensity were weak.
Fig. 3 (A)Promoter activity of pAnb1, pRox1 and constitutive(pCyc) measured by means of Fluorescence of each cell. (B)Fluorescence distribution of cells in pAnb1 strain. (C)Fluorescence distribution of cells in pRox1 strain.(D)Fluorescence distribution of cells in Constitutive(pCyc) strain.
Discussion
As can be seen from the experimental results, both pAnb1 and pRox1 showed a promoter activity increase with the change of oxygen concentration. However, the increase in activity was relatively small in pRox1, and also the change in activity was observed in the constitutive promoter(pCyc:BBa_I766555). For the explanation of the results, two possibility can be thought:
Possibility 1: The decrease in expression level is caused by a decrease in cellular metabolic activity under anaerobic conditions.
Possibility 2: The pCyc used as a constitutive promoter may be oxygen-responsive. (Some paper reported that pCyc have oxygen-dependent expression change [5])
As a future prospect, we need to set up an experiment to re-evaluate these possibilities. And also, if possibility 1 actually makes a significant difference, we can build a system that directly uses this nature of yeast cells.
Defensin Secretion
Overview
Goal
To construct a system that prevents infection of wounds and keeps them clean by making Saccharomyces cerevisiae secrete HBD3 (human beta-defensin-3), an antimicrobial protein.
Result
A yeast strain that secretes HBD3 was developed, and its effect on the yeast itself was evaluated. Antimicrobial assays were also performed by using its culture supernatant. A yeast growth assay showed that HBD3 secretion affected the growth rate of yeast but did not affect the final cell density. This antimicrobial assay qualitatively suggested that HBD3 had antibacterial activity against E. coli. However, the results lacked certainty and required additional experiments.
Future Direction
We are going to qualify the secretion of HBD3 and reconsider which promoter to use. We will also test whether it shows antimicrobial activity when yeast is incorporated into a cell fiber.
System Design
Among the various human antimicrobial proteins, HBD3 is known for its effectiveness against both Gram-negative and Gram-positive bacteria, independent of the environmental salt concentration [6]. Therefore, we chose HBD3 for this project.
Parts Construction
Based on the design described above, the plasmid was constructed by assembling a linear backbone and the sequence containing HBD3 (CYC1 promoter-HBD3-ADH1 terminator) using SLiCE. The backbone was linearized at both ends of MCS of BBa_K1680016, and the fragment including HBD3 was synthesized in IDT. The desired yeast strain was established by transforming the plasmid into yeast.
The specific design was to incorporate the sequence CYC1 promoter-HBD3-ADH1 terminator into a plasmid and make it work in yeast.
Effect on the Yeast
We constructed an HBD3-secreting yeast strain following the procedure described above and evaluated its growth characteristics. It was reported that HBD3 has antimicrobial activity against yeast [6], and secretion of HBD3 may affect the growth of yeast. Therefore, the growth rate between the HBD3-secreting strain and the control strain was compared in our project (n=3).
The results of the OD600 measurement of HBD3-secreting yeast were shown in Figure 4. The control strain was the pRS316 transformed one (BBa_K1680016 compatible vector without insert). As shown in Figure 4, HBD3 dramatically affected yeast growth during the exponential phase. This property was observed particularly at 330 minutes after the inoculation. However, since the final concentration was almost the same, HBD3 secretion was considered to have little effect on the number of yeast in the end. Thus, this strain is expected to be sufficient for practical implementation.
Fig. 4 The growth curve of the HBD3-secreting strain and the control (pRS316).
Antibacterial Assay
Dressings were soaked with supernatant from the culture of HBD3-secretion strain and the control strain, and then they were placed on the plates coated with E. coli. The plates were incubated at 37℃ for 48 hours. As shown in Figure 5, a halo formed around the dressing soaked with HBD3-secretion strain culture supernatant, which was not observed with the control. The results indicated that HBD3 had antibacterial activity against E. coli.
However, this experiment was conducted with n=1, and the results were not reproducible. It is necessary to experiment with more samples to measure the antibacterial activity of HBD3 more accurately. (Antimicrobial assay of our yeast supernatant was also conducted by team Kyoto, and it brought similar results. Please check here.)
Fig. 5 The antibacterial activity of the HBD3-secreting strain. The red arrow indicates the halo.
Pathogen Detection
Overview
Goal
To construct an infection detection system by detecting quorum sensing signals of Pseudomonas aeruginosa using S. cerevisiae.
Result
We planned to construct the desired plasmids by assembling six DNA fragments. We successfully created DNA fragments consisting of three segments and two segments, but we did not have enough time to complete the whole construction of plasmids.
Future Direction
After completing the construction of the plasmids and transferring them into yeast, we are going to examine the threshold of infection detection ability by testing the response to various concentrations of AHL. In addition, by changing or mutating the promoters used, we will adjust the threshold.
System Design
The aim is to construct a system to detect quorum sensing signals by modifying the quorum sensing pathway of prokaryotes and incorporating it into S. cerevisiae, but the first problem was to decide which path to use. We chose LasR and QscR proteins, which detect 3OC12-HSL [7][8], an AHL that plays a central role in quorum sensing in P. aerugionosa. We also decided to use the LuxR protein pathway since the Tsinghua University iGEM team 2013 had demonstrated the detection of a quorum sensing signal of P. aerugionosa with LuxR [9]. In incorporating the prokaryotic system into eukaryotes, we designed the plasmids based on the Tsinghua University 2013 project, as described in the Description page (Fig. 6).
Fig. 6 The design of the plasmid.
Plasmid Construction
Design
The DNA fragments shown in Table 3 were prepared by ordering them from IDT.
Table3. The details of the prepared fragments.
| Fragment name | Included regions or parts |
|---|---|
| fragment1 | pTEF & Flag |
| fragment2 | LuxR/LasR/QscR, pLux/pLas/pQsc, ADH1 terminator, & cyc 100 mini promoter |
| monoVP16 | VP16 |
| backbone | mCherry, CYC1 terminator, & pRS316 |
The specific plasmid construction plan is described below (Fig. 7).
Step1: To splice fragment1 and mono VP16
Step2: To splice two pieces of monoVP16
Step3: To splice fragment1+monoVP16 and monoVP16+monoVP16
Step4: To splice fragment2 and the fragment constructed in Step3
Step5: To splice backbone and the fragment constructed in Step4 by SLiCE
Fig. 7 The initial plasmid construction plan.
Results
Step1 was successful, but Step2 did not go well, and smear bands were obtained after PCR (Fig. 8).
Fig. 8 The results of PCR of Step1 (right lane) and Step2 (left lane).
Redesign
Based on the results, the order of the plasmid assembly was changed: the construction was kept without tandem repeats at the initial steps because the tandem repeats could cause various by-products during PCR. The redesigned construction procedure was as follows (Fig. 9).
Step1: To splice fragment1 and monoVP16
Step2: To splice monoVP16 and fragment2
Step3: To splice backbone and the fragment constructed in Step1
Step4: To splice the fragments constructed in Step2 and Step3
Step5: To splice monoVP16 and the fragment constructed in Step4 by SLiCE
Fig. 9 The redesigned plasmid construction method.
Results
Step1, Step2, and Step3 were successful, but Step4 did not work (Fig. 10).
Fig. 10 The results of PCR of Step2 (A), Step3 (B), and Step4 (C). For Step4, since Lux, Las, and Qsc (from right to left) were used for fragment2, there were three lanes.
We also tried to assemble the products of Step2, Step3, and monoVP16 by SLiCE, but no desired E. coli strain was obtained (Fig. 11).
Fig. 11 The results of PCR on E. coli colonies which could have the desired plasmid.
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