iGEM 2025 — Team csu-china

# Lab Note

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## Table of Contents

* [Overview](#Overview)
* [Result](#Result)
* [Summary](#Summary)
* [References](#references)

# LAB Work

Engineering success

## Overview

Our project aims to design a highly responsive lactate sensor and reduce the high lactate concentration in the tumor microenvironment (TME) to achieve therapeutic effects. To this end, our system is divided into two components: a lactate-sensing module (Lactate Sensor) and a lactate-degrading module (Lactate Oxidase). Through multiple rounds of construction and experimental validation, we have successfully developed a highly responsive lactate sensor that can also function to degrade extracellular lactate, thereby achieving the therapeutic goal.

## Results

Design of the Lactate-Sensing Module

First Construction of the Lactate-Sensing Module

We assembled the selected components (see the main text of the Wiki for details). Referring to the design concept of the "split-TEV" system in the literature, we split the TEV protease into N-terminal and C-terminal fragments, which were then linked to the N-terminus and C-terminus of the LIdR protein (A protein for sensing lactate) respectively, completing the first-generation design of our sensor, designated as LS1.0. However, it failed to achieve the desired performance (Figure 1).

![ff1](https://static.igem.wiki/teams/5731/lab/eng1.avif)

**Figure 1.** This figure presents the experimental results of the LS1.0 sensor verification using the dual-luciferase reporter gene assay. The ordinate (RLU, relative light unit) reflects luciferase enzyme activity; the results show that even without lactate stimulation (0mM), the TEV enzyme activity of the LS1.0 group is still high, indicating background activity issues in the first-generation sensor.RLU represents the ratio of firefly luciferase fluorescence intensity to Renilla luciferase fluorescence intensity. Each group included 3 biological replicates, and the data are presented as mean ± Standard Deviation (SD). Data were analyzed using one-way analysis of variance (ANOVA). "ns" indicates no significant difference compared with the control group, while "\*" indicates a significant difference with p < 0.05 relative to the control group.

Second Construction of the Lactate-Sensing Module

After team discussions, we attributed this issue to the excessively short physical distance between the split TEV protease fragments on the LIdR protein, which caused the TEV protease to become active and function even before lactate binding. Therefore, we made an improvement: we split the LIdR protein into two parts (N-terminal and C-terminal), which were then linked to the N-terminal and C-terminal of the TEV protease respectively, resulting in our LS2.0. We conducted experiments with it, but unfortunately, the results were still unsatisfactory (Figure 2).

![ff2](https://static.igem.wiki/teams/5731/lab/eng2.avif)

**Figure2.** This figure presents the experimental results of the LS2.0 sensor verification using the dual-luciferase reporter gene assay. The ordinate (RLU) represents TEV enzyme activity; similar to LS1.0, the results indicate that the LS2.0 group still exhibits high TEV enzyme activity in the absence of lactate (0mM), meaning the background activity problem was not resolved.RLU represents the ratio of firefly luciferase fluorescence intensity to Renilla luciferase fluorescence intensity. Each group included 3 biological replicates, and the data are presented as mean ± Standard Deviation (SD). Data were analyzed using one-way analysis of variance (ANOVA). "ns" indicates no significant difference compared with the control group.

Third Construction of Lactate-Sensing Module

We observed that the LS2.0 still exhibited high TEV protease activity in the absence of lactate. To address this, we decided to further optimize the sensor based on the aforementioned design. In this iteration, we abandoned the fixed linkage pattern of connecting the N-terminus of LIdR to the N-terminus of TEV and the C-terminus of LIdR to the C-terminus of TEV. Instead, we altered the N/C-terminal connection modes between the two proteins and constructed 8 different combinations (see the main text of the Wiki for details).（Figure 3）

![f3](https://static.igem.wiki/teams/5731/lab/eng3.avif)

**Figure3.** This figure provides a visual diagram of the structural composition of each sensor in the LS3.1-LS3.8 series. It clearly marks the connection relationship between Lac-N/Lac-C and TEV-N/TEV-C in each sensor, serving as a design basis for subsequent background screening.

Subsequently, we proceeded to conduct further exploration on these 8 sensor combinations and successfully identified the optimal combination for the lactate sensor.

Based on the summary of failure causes from the two previous screenings, we first conducted a background screening in this iteration (Figure 4). Subsequently, we performed multiple rounds of repeated verification on the three combinations with low background, and finally confirmed that LS3.5 was the sensor with high sensitivity and stability we required (Figure 5).

![ff4](https://static.igem.wiki/teams/5731/lab/eng4.avif)

**Figure4.** This figure presents the background screening results of the LS3.1-LS3.8 sensors. The ordinate (RLU) reflects the TEV enzyme activity of each sensor without lactate stimulation; the results help screen out sensors with low background activity (e.g., LS3.5, LS3.6, LS3.8).RLU represents the ratio of firefly luciferase fluorescence intensity to Renilla luciferase fluorescence intensity. Each group included 3 biological replicates, and the data are presented as mean ±Standard Deviation (SD).

![ff5](https://static.igem.wiki/teams/5731/lab/eng5.avif)

**Figure5.** This figure shows the sensitivity and stability verification results of the screened low-background sensors (LS3.5, LS3.6, LS3.8) under different lactate concentrations (0mM, 1mM, 5mM). The ordinate (RLU) indicates TEV enzyme activity; the results confirm that LS3.5 has the highest sensitivity and stability, as it shows a more obvious response to lactate concentration changes while maintaining low background.RLU represents the ratio of firefly luciferase fluorescence intensity to Renilla luciferase fluorescence intensity. Each group included 3 biological replicates, and the data are presented as mean ± Standard Deviation (SD). Data were analyzed using one-way analysis of variance (ANOVA). "ns" indicates no significant difference compared with the control group, while "\*" indicates a significant difference with p < 0.05 relative to the control group.

Design for Lactate Degradation

Design of the sLOx Enzyme

We selected lactate oxidase (LOx) as our tool enzyme. However, given that LOx is a cytoplasmic enzyme, we modified it to enable secretion outside the cell. Specifically, we added a signal sequence of rat follicle-stimulating hormone beta subunit (rat FSHB) to the N-terminus of the LOx sequence (Figure 6) to facilitate the secretion of the LOx enzyme out of the cell.

![ff6](https://static.igem.wiki/teams/5731/lab/eng6.avif)

**Figure6.** This figure shows the structural design of the engineered secreted lactate oxidase (sLOx). It illustrates that a signal sequence derived from the rat follicle-stimulating hormone β-subunit (rat FSHB) was fused to the N-terminus of the lactate oxidase (LOx) sequence, and a FLAG tag was also included in the structure (with the total length marked as approximately 1250 base pairs). The rat FSHB signal sequence is responsible for directing the newly synthesized sLOx protein into the cell secretory pathway, ensuring that sLOx is secreted extracellularly to exert its lactate-degrading function, rather than being retained in the cytoplasm.

This is the secreted lactate oxidase (sLOx) we designed.

Secretion and Functional Verification of the sLOx Enzyme

We first conducted a secretion assay for the designed sLOx enzyme, and ultimately confirmed via Western blot that it could be secreted into the Culture Medium (Figure 7).

![ff7](https://static.igem.wiki/teams/5731/lab/eng7.avif)

**Figure7.** This figure displays the results of the Western blot assay used to verify sLOx protein secretion. The blot includes two sample types: culture medium (extracellular fluid) and cell lysate (intracellular fluid), with both NC (negative control) and LS3.5 (sensor-sLOx system) groups tested. The presence of the sLOx protein band in the culture medium of the LS3.5 group (while no obvious band is detected in the NC group's culture medium) confirms that the engineered sLOx protein was successfully secreted from 293T cells into the extracellular space.

Subsequently, we conducted experiments on the extracellular lactate degradation by sLOx protein. We combined the optimized lactate sensor (screened out in previous steps) with the designed sLOx, which constitutes our LS3.5 lactate-responsive system. The lactate-degrading function of this system was verified by detecting the lactate content in the Culture Medium.(Figure 8)

![ff8](https://static.igem.wiki/teams/5731/lab/eng8.avif)

**Figure8.** This figure shows the results of the extracellular lactate degradation assay. The ordinate represents the relative concentration of extracellular lactate, and the abscissa shows the NC group and the LS3.5 group (sensor-sLOx system group). The results clearly indicate that compared with the NC group, the extracellular lactate concentration in the LS3.5 group is significantly lower, directly demonstrating that the secreted sLOx protein has a stable and efficient lactate-degrading function, which can reduce the local extracellular lactate concentration as expected.

## Summary

In the process of constructing our lactate-responsive system, we performed three rounds of engineering for the lactate-sensing module. Throughout these three rounds, we continuously optimized the sensor; ultimately, during the third round of engineering, we obtained the lactate-sensing combination with the optimal performance—LS3.5—through experimental validation. Subsequently, we combined LS3.5 with our designed secreted lactate oxidase (sLOx) to construct our lactate-responsive system. Experimental validation further confirmed that our system is capable of both sensing lactate and degrading lactate.

With this, the construction of our lactate-responsive system was successfully accomplished!

## References

1. Wintgens JP, Rossner MJ, Wehr MC. Monitoring activities of receptor tyrosine kinases using a universal adapter in genetically encoded split TEV assays. Cell Mol Life Sci. 2019;76(6):1185-1199. doi:10.1007/s00018-018-03003-2
2. Li X, Zhang Y, Xu L, Wang A, Zou Y, Li T, Huang L, Chen W, Liu S, Jiang K, Zhang X, Wang D, Zhang L, Zhang Z, Zhang Z, Chen X, Jia W, Zhao A, Yan X, Zhou H, Zhu L, Ma X, Ju Z, Jia W, Wang C, Loscalzo J, Yang Y, Zhao Y. Ultrasensitive sensors reveal the spatiotemporal landscape of lactate metabolism in physiology and disease. Cell Metab. 2023;35(1):200-211.e9. doi:10.1016/j.cmet.2022.10.002
3. Meister SW, Parks L, Kolmar L, Borras AM, Ståhl S, Löfblom J. Engineering of TEV protease variants with redesigned substrate specificity. Biotechnol J. 2023;18(11):e2200625. doi:10.1002/biot.202200625
4. Aguilera L, Campos E, Giménez R, Badía J, Aguilar J, Baldoma L. Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. J Bacteriol. 2008;190(8):2997-3005. doi:10.1128/JB.02013-07
5. Faubert B, Li KY, Cai L, Hensley CT, Kim J, Zacharias LG, Yang C, Do QN, Doucette S, Burguete D, Li H, Huet G, Yuan Q, Wigal T, Butt Y, Ni M, Torrealba J, Oliver D, Lenkinski RE, Malloy CR, Wachsmann JW, Young JD, Kernstine K, DeBerardinis RJ. Lactate Metabolism in Human Lung Tumors. Cell. 2017;171(2):358-371.e9. doi:10.1016/j.cell.2017.09.019
6. Haas R, Smith J, Rocher-Ros V, Nadkarni S, Montero-Melendez T, D'Acquisto F, Bland EJ, Bombardieri M, Pitzalis C, Perretti M, Marelli-Berg FM, Mauro C. Lactate Regulates Metabolic and Pro-inflammatory Circuits in Control of T Cell Migration and Effector Functions. PLoS Biol. 2015;13(7):e1002202. doi:10.1371/journal.pbio.1002202 doi:10.1371/journal.pbio.1002202
7. Andre C, Kim SW, Yu XH, Shanklin J. Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H2O2 to the cosubstrate O2. Proc Natl Acad Sci U S A. 2013;110(8):3191-3196. doi:10.1073/pnas.1218769110
8. Li XY, Wang XH, Huang XQ, Wu TT, Xu W, Wu SS. Development of a cell-based peroxisome proliferator-activated receptors (PPARs) screening model and its application for evaluation of triterpenoids isolate from Alismatis Rhizoma. Zhongguo Zhong Yao Za Zhi. 2016;41(21):4015-4022. doi:10.4268/cjcmm20162121

## Table of Contents

* [1. Experimental Protocol](#aaa)

+ [1.1 Plasmid](#bbb)
+ [1.2 Cell Culture](#fff)
+ [1.3 Western Blot](#ppp)
+ [1.4 Lactate Determination](#uuu)

* [2. References](#references)

# LAB Work

Experiment Protocol

## Experimental Protocol

1. Plasmid

1.1 Plasmid Construction

The inserted DNA for all plasmids except pGL4.35 were synthesized and cloned into respective vectors by Sangon corporation.

1.2 Plasmid Amplification

(1) Retrieve competent DH5α Escherichia coli cells from the -80°C freezer and thaw them on ice (do not vortex or pipette vigorously).

(2) Remove the plasmids to be amplified from the -20°C freezer and place them on ice for thawing.

(3) Label microcentrifuge tubes with the respective plasmid names.

(4) Add 30 μL of DH5α Escherichia coli to each labeled microcentrifuge tube.

(5) Add 1 μL of each thawed plasmid to the corresponding tube, then return the plasmids to the -20°C freezer immediately after use.

(6) Incubate the tubes containing the mixture on ice for 30 minutes.

(7) Transfer the tubes to a preheated 42°C heat block for a 90-second heat shock, then immediately place them back on ice for incubation for at least 2 minutes.

(8) Remove ampicillin from the -20°C freezer and thaw by warming in hand.

(9) Retrieve LB (-) medium (sterile) from the 4°C refrigerator. Prepare a test tube rack, alcohol lamp, and culture tubes (pre-labeled with the corresponding plasmid names). Ignite the alcohol lamp, and under aseptic conditions near the flame, add 5 mL of LB (-) medium and 5 μL of ampicillin to each culture tube.

(10) Remove the ice-incubated plasmid mixtures and transfer the entire volume into the corresponding labeled culture tubes (do not fully tighten the caps).

(11) Place the 6 culture tubes on a shaking incubator and incubate at 37°C with vigorous shaking (200–250 rpm) for 12–16 hours.

1.3 Plasmid Extraction

(1) Label 15 mL centrifuge tubes with the names of the plasmids to be extracted.

(2) Retrieve the culture tubes from the shaker and transfer the bacterial cultures to the corresponding 15 mL centrifuge tubes. Centrifuge at 13,000 rcf for 1 minute (ensure symmetrical placement in the centrifuge, same for subsequent steps).

(3) Remove the centrifuged tubes, carefully discard the supernatant, and resuspend the bacterial pellet by gentle pipetting with the residual medium.

(4) Label microcentrifuge tubes with the names of the plasmids to be extracted, and transfer the resuspended bacterial solution to the corresponding tubes.

(5) Centrifuge the microcentrifuge tubes at 13,000 rcf for 1 minute. After centrifugation, remove the tubes and discard the supernatant.

(6) Add 250 μL of Buffer P1 (supplemented with RNase A, retrieved from 4°C storage) to each tube, then resuspend the bacterial pellet by vortexing at maximum speed until no clumps remain.

(7) Add 250 μL of Buffer P2 to the resuspended solution and invert the tube 8–10 times until the solution becomes clear.

(8) Add 350 μL of Buffer P3 to each tube and immediately invert 8–10 times.

(9) Centrifuge the tubes at 13,000 rcf for 10 minutes.

(10) Place a HiPure DNA Mini Column Ⅳ (hereinafter referred to as "column") into a collection tube. Carefully transfer the supernatant to the column and centrifuge at 13,000 rcf for 60 seconds.

(11) Discard the filtrate, return the column to the collection tube, add 500 μL of Buffer PW1 to the column, and centrifuge at 13,000 rcf for 60 seconds.

(12) Discard the filtrate, return the column to the collection tube, add 600 μL of Buffer PW2 (pre-diluted with absolute ethanol) to the column, and centrifuge at 13,000 rcf for 60 seconds.

(13) Discard the filtrate, return the column to the collection tube, add 300 μL of Buffer PW2 (pre-diluted with absolute ethanol) to the column, and centrifuge at 13,000 rcf for 60 seconds.

(14) Discard the filtrate, return the column to the collection tube, and centrifuge at 13,000 rcf for 60 seconds to dry the column.

(15) Place the column into a sterile 1.5 mL microcentrifuge tube. Add 50 μL of Elution Buffer or sterile water to the center of the column membrane. Allow to stand for 1 minute, then centrifuge at 13,000 rcf for 60 seconds to elute the plasmid DNA.

(16) Determine the concentration and purity of the extracted plasmid DNA using a microvolume spectrophotometer: load 1 μL of the eluted sample, and the instrument-connected software will generate quantitative data for nucleic acid concentration (ng/μL) and A260/A280 ratio.

2. Cell Culture

2.1 HEK293T Cell Culture

2.1.1 Cell Resuscitation

(1) Put the cryopreservation tube into a 37°C water bath to thaw, and take out the cryopreservation tube immediately when there is only a small amount of cell ice crystals left in the tube.

(2) Transfer the cell suspension to a 15 mL centrifuge tube, add 9 times the volume of 10% DMEM cell culture medium, and centrifuge at 1,000 rpm for 5 minutes.

(3) Remove the supernatant, add an appropriate amount of 10% DMEM cell culture medium to resuspend the cells, then transfer them to a culture dish, and add an appropriate amount of 10% DMEM cell culture medium. Gently shake the culture dish quickly in the front-back and left-right directions several times.

(4) Place the culture dish in a 37°C cell incubator containing 5% CO₂for culture.

2.1.2 Cell Passage and Culture

(1) Perform passage operation when the cell density in the culture dish is observed to be 80%-90%.

(2) Discard the supernatant in the culture dish, add an appropriate amount of trypsin to digest the cells. When a large number of cells are observed to fall off, add 3 times the volume of 10% DMEM to stop the digestion, and gently pipette to detach the cells.

(3) Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1,000 rpm for 3 minutes.

(4) Discard the supernatant, add an appropriate amount of 10% DMEM cell culture medium to resuspend the cells according to the experimental needs, and distribute the cell suspension into culture dishes or well plates, then place them in the cell incubator.

(5) Observe the state of the cells and the culture medium in the culture dish every day, and perform medium change operation in a timely manner.

2.1.3 Cell Cryopreservation

(1) Aspirate and discard the original medium, add an appropriate amount of trypsin to digest the cells, add 3 times the volume of 10% DMEM to pipette and detach the cells. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 800 rpm for 5 minutes.

(2) Discard the supernatant, add 1.5 mL of serum-free cell cryopreservation solution to resuspend the cells.

(3) Transfer the cell suspension to a cryopreservation tube, clearly write the information, place it in a -80°C refrigerator for 24 hours, and then transfer it to a liquid nitrogen tank for long-term storage.

2.2 Dual-Luciferase Reporter Gene Assay

（1）Take out the 293T cells cultured in the 24-well plate from the incubator, aspirate the medium, and wash once with 100 µL of 1×PBS.

（2）Add 100 µL of 1×PBS to each well.

（3）Add 100 μL of 1×Reporter Lysis Buffer (diluted with 5×Reporter Lysis Buffer and ddH₂O).

（4）Pipette gently multiple times to transfer to a 1.5 mL EP tube, and mark it well.

（5）Vortex and let stand for 5 minutes.

（6）Measure the luciferase value

① Set up the FB12 Sirius software, select Dual Assay for Protocol types, select Dual-Luciferase Assay for Runnable protocols, and click Run to start the experiment.

② Pipette repeatedly to mix well, then put it into the Sivius single-tube chemiluminescence detector for detection. The detector will automatically detect the intensity of fluorescence, which will be displayed numerically on the FB12 Sirius software, and the first data will be obtained from the measurement.

③ Add 10 µL of renilla and 1 µL of terminator mixture to each tube (prepared freshly before use, and the stock solution is stored at -80°C). Pipette to mix well, then put it into the Sivius single-tube chemiluminescence detector for detection, and the second data will be obtained from the measurement.

④ Copy all data, export it to an Excel spreadsheet, and perform normalization processing and analysis on the data using Stop&Glo data.

3. Western Blot

3.2.1 Preparation of 12% SDS-PAGE Gel

(1)Rinse the glass plates thoroughly, followed by a final rinse with ddH₂O, and allow them to air-dry at room temperature. Once dry, carefully align a set of glass plates tightly and fix them on a dedicated gel casting stand.

(2)Prepare the separating gel by sequentially adding the reagents listed in the table below into a 50 ml centrifuge tube:

| **Reagents** | **Volume** |
| --- | --- |
| ddH20 | 3.3ml |
| 30%Methylene Bisacrylamide | 4.0ml |
| Tris-HCl(pH8.8) | 2.5ml |
| 10%APS | 10ul |
| 10%SDS | 10ul |
| TEMED | 4ul |

After repeatedly inverting and mixing gently, slowly add approximately 7 mL of the mixture into the gap between the glass plates (take care to minimize bubble formation). Then carefully add 2 mL of absolute ethanol to seal the gel—at this point, a distinct boundary layer can be observed between the absolute ethanol and the separating gel.

(3)Allow the mixture to stand at room temperature until the separating gel solidifies. Discard the absolute ethanol layered on top of the gel, then invert the glass plate assembly onto filter paper to dry.

(4)Prepare the stacking gel by sequentially adding the reagents listed in the table below into a 50 mL centrifuge tube：

| **Reagents** | **Volume** |
| --- | --- |
| ddH20 | 2.1ml |
| 30%Methylene Bisacrylamide | 0.5ml |
| Tris-HCl(pH8.8) | 0.38ml |
| 10%APS | 30ul |
| 10%SDS | 30ul |
| TEMED | 3ul |

After mixing the solution thoroughly, add 3 mL of it into the gap above the separating gel in the glass plate assembly, then gently insert a cleaned gel comb into the stacking gel.

After allowing the gel to solidify at room temperature, remove the SDS-PAGE gel from the gel casting stand. Carefully pull out the gel comb, rinse off any residual gel on the surface of the glass plates with running water, and use the gel immediately or store it in a sealed bag at 4℃.

3.2.2 Protein Elect SDS-PAGE Electrophoresis and Development

(1) Fix the SDS-PAGE gel in the corresponding position of the electrophoresis tank, fill it with freshly prepared electrophoresis buffer, and ensure the liquid level covers the upper edge of the gel.

(2) Add an appropriate volume of protein Marker and protein loading buffer into the gel wells in sequence, turn on the power, cover the electrophoresis tank lid, and perform electrophoresis at a constant voltage of 90V for 30 minutes.

(3) At this point, prepare the transfer buffer in advance (freshly prepared and used immediately), and pre-cool it in a 4°C or -20°C refrigerator.

(4) Subsequently, switch to a constant voltage of 120V and continue electrophoresis for approximately 60 minutes. The electrophoresis time can be adjusted according to the real-time position of the corresponding bands of the protein Marker and the molecular weight of the target protein.

(5) After electrophoresis is completed, open the transfer夹板 and place it in a tray, pour the pre-cooled transfer buffer into an enamel tray, take out the SDS-PAGE gel from the electrophoresis tank, gently pry open the glass plate with a plastic pry plate, and take out the gel block and place it on the filter paper near the black side of the transfer clamp.

(6) Briefly soak the cut PVDF membrane in methanol to activate the positive charge groups on the membrane, then lay it flat over the gel block, and use a plastic pry plate to drive out air bubbles between the membrane and the gel block.

(7) Cover with filter paper and sponge in sequence, use a plastic pry plate to drive out air bubbles again, close the transfer clamp and clamp it tightly to form a "sandwich" structure of "transfer clamp transparent side - sponge - filter paper - PVDF membrane - PAGE gel - filter paper - sponge - transfer clamp black side".

(8) Insert the transfer clamp into the transfer tank in the direction of "black to black" (the black side of the transfer clamp is aligned with the black side of the transfer tank), put in the ice grid, fill it with fresh transfer buffer, place the transfer tank in an ice-water mixture bath, and cover the transfer tank lid.

(9) Turn on the power, with a constant current of 290mA, and transfer for 90 minutes.

(10) After the transfer is completed, open the transfer clamp to take out the PVDF membrane, with the side close to the gel block facing up, mark the upper right corner, put it into the incubation box, pour 5% skimmed milk, and seal it on a shaker at room temperature for 1 hour with slow shaking.

(11) After sealing, recover the skimmed milk, add an appropriate amount of 0.1% TBST membrane washing solution, shake quickly on the shaker for 5 minutes, discard the membrane washing solution, and repeat 3 times to rinse off the residual milk.

(12) Pour in the corresponding primary antibody, place it on a shaker in a 4°C cold storage, and incubate overnight with slow shaking (the antibody incubation time can be adjusted according to experimental experience).

(13) After the primary antibody incubation is completed, recover the primary antibody, first quickly rinse the residual primary antibody in the box with membrane washing solution, then add an appropriate amount of membrane washing solution, and wash the membrane on the shaker with quick shaking for 8 minutes each time, a total of three times.

(14) Add the corresponding secondary antibody incubation solution (prepared with 5% skimmed milk) according to the species source of the primary antibody, and incubate on a shaker at room temperature with slow shaking for 1 hour.

(15) After incubation, discard the secondary antibody, add an appropriate amount of membrane washing solution, shake quickly on the shaker for 8 minutes, and wash the membrane three times.

(16) Pipette equal volumes of developing solution A and B into a 1.5mL EP tube, mix well, drop the developing solution evenly on the membrane to cover the entire membrane, and after incubating for an appropriate time, perform development with a multi-functional imager.

(17) The development results are scanned and analyzed with ImageJ or Photoshop software. After measuring the grayscale value of the target band, first normalize it with the grayscale value of the internal reference, and then compare the changes in the expression level of the target protein.

4.Lactate Determination

**(1)Preparation of cell culture supernatant samples:** For adherent cells, directly aspirate the culture supernatant for subsequent determination; for suspension cells, aspirate the culture supernatant after centrifugation for subsequent determination.

**(2)Preparation of the assay kit:**

a. Thaw BeyoLysis™ Buffer A for Metabolic Assay and Lactate Assay Buffer, equilibrate them to room temperature, then mix well for later use. Store other reagents on ice for subsequent use, and immediately store them under the conditions required by the kit after use.

b. Preparation of WST-8 Chromogenic Working Solution: Prepare an appropriate amount of chromogenic working solution based on a volume of 50 µl per detection reaction. Mix 44 µl of Lactate Assay Buffer, 2 µl of Enzyme Solution, 2 µl of Substrate, and 2 µl of WST-8 thoroughly to prepare 50 µl of WST-8 chromogenic working solution. Prepare an appropriate volume of WST-8 chromogenic working solution according to the number of samples to be tested (including standards), with specific preparation methods referring to the table below. The prepared WST-8 chromogenic working solution can be used on the same day if stored at 4°C or on ice in the dark, but it is recommended to prepare it freshly as needed.

| **Samples** | **1** | **10** | **20** | **50** |
| --- | --- | --- | --- | --- |
| Lactate Assay Buffer (μl) | 44 | 440 | 880 | 2200 |
| Enzyme Solution (μl) | 2 | 20 | 40 | 100 |
| Substrate (μl) | 2 | 20 | 40 | 100 |
| WST-8 (μl) | 2 | 20 | 40 | 100 |
| WST-8 Working Solution (µl) | 50 | 500 | 1000 | 2500 |

**Note 1:** Since the amount of enzyme solution used is small and it is prone to sedimentation, it is necessary to gently centrifuge it before use, and then mix it properly before use.

**Note 2:** The presence of NADH, NADPH, etc. will interfere with the detection of lactate. If the sample contains NADH or NADPH, a background control well for the sample should be set up simultaneously, and the chromogenic working solution without the enzyme solution should be added. That is, when preparing the WST-8 chromogenic working solution, 2 µl of Enzyme Solution should be replaced with Lactate Assay Buffer. When calculating, the reading value of the sample well needs to subtract the reading value of the background control well.

**(3)Setup of L-Lactate Standard Curve:**Take 1 µl of L-Lactate Standard (100 mM), add 199 µl of BeyoLysis™ Buffer A for Metabolic Assay, mix well to prepare 200 µl of L-Lactate Standard with a concentration of 0.5 mM. Pipette 0, 2, 5, 10, 20, and 50 µl of 0.5 mM L-Lactate Standard respectively into the standard wells of a 96-well plate, and make up to 50 µl with the corresponding BeyoLysis™ Buffer A for Metabolic Assay. At this point, the L-lactate concentration and molar amount in each well of the standard curve are 0, 0.02, 0.05, 0.1, 0.2, 0.5 mM and 0, 1, 2.5, 5, 10, 25 nmol, respectively.

**(4)Sample Determination:**

a.Pipette 50 µl of the sample prepared in step 1 into the sample wells of a 96-well plate. Meanwhile, set up wells containing only BeyoLysis™ Buffer A for Metabolic Assay as blank control wells.

Note: The original solution is diluted 50-fold here.

b.Add 50 µl of WST-8 chromogenic working solution to each well and mix well. Incubate at 37°C in the dark for 30 minutes.

c.Measure the absorbance at 450 nm.

d.Subtract the signal value of the zero-concentration standard well from the signal values of each standard curve well. Establish a standard curve and calculate the concentration of lactate in the sample (A). The signal value of the sample well should be subtracted by that of the blank control well. If the signal of the sample background control well is relatively high, the signal value of the sample needs to be subtracted by that of the sample background control. A good linear relationship exists within the concentration range of 0.02-0.5 mM.

**(5)Data processing:**

a. First, import the measured data into EXCEL for preliminary data processing, that is, use the data at 0 mM of the standard curve to zero the measured data.

b. Divide the processed data by the slope k value of the obtained standard curve to get the measured lactate concentration.

c. Multiply the measured lactate concentration by the dilution factor (50) to obtain the final result.

d. Import the finally processed data into Prism for plotting. The abscissa is the grouping information of each group of data, and the ordinate is the relative lactate concentration of each group of data. Draw a bar chart to analyze the difference in relative lactate concentration among groups.

The calculation formula for lactate concentration is as follows: C (mM) = A × n

Note 1: A is the diluted sample lactate concentration (mM) determined according to the standard curve in step 4d; n is the sample dilution factor in step (4)a.

Note 2: The calculated lactate concentration includes the molar concentration of lactate and L-lactate, which can also be understood as including the molar concentration of lactate and L-lactate salt. The above is only for the convenience of expression and is only described as lactate. If necessary, the mass concentration (μg/ml) of L-lactate in the sample can be calculated according to the molecular weight of L-lactate, 89.07, as C×0.08907.

## References

1. Wintgens JP, et al. Monitoring activities of receptor tyrosine kinases using a universal adapter in genetically encoded split TEV assays. Cellular and Molecular Life Sciences. 2019;76:1185-1199. doi:10.1007/s00018-018-03003-2
2. Li X, et al. Ultrasensitive sensors reveal the spatiotemporal landscape of lactate metabolism in physiology and disease. Cell Metabolism. 2023;35:200-211.e9. doi:10.1016/j.cmet.2022.10.002
3. Markert CL. Cell Biochem Funct. 1984;2(3):131-134.
4. Feng Y, et al. Cancer Med. 2018;7(12):6124-6136.
5. Cristescu ME, Innes DJ, Stillman JH, Crease TJ. BMC Evol Biol. 2008;8:268.
6. Forkasiewicz A, et al. Cell Mol Biol Lett. 2020;25:35.
7. Claps G, et al. Nat Rev Clin Oncol. 2022;19(12):749-762.
8. Aguilera L, Campos E, Giménez R, Badía J, Aguilar J, Baldoma L. Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. J Bacteriol. 2008;190(8):2997-3005. doi:10.1128/JB.02013-07
9. Faubert B, et al. Lactate Metabolism in Human Lung Tumors. Cell. 2017;171(2):358-371.e9. doi:10.1016/j.cell.2017.09.019
10. Kapust RB, Waugh DS. Controlled intracellular processing of fusion proteins by TEV protease. Protein Expr Purif. 2000;19(2):312-318. doi:10.1006/prep.2000.1251
11. Meister SW, et al. Engineering of TEV protease variants with redesigned substrate specificity. Biotechnol J. 2023;18:e2200625. doi:10.1002/biot.202200625
12. Wehrman T, Kleaveland B, Her JH, Balint RF, Blau HM. Protein-protein interactions monitored in mammalian cells via complementation of beta -lactamase enzyme fragments. Proc Natl Acad Sci U S A. 2002;99(6):3469-3474. doi:10.1073/pnas.062043699
13. Rossi F, Charlton CA, Blau HM. Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc Natl Acad Sci U S A. 1997;94(16):8405-8410. doi:10.1073/pnas.94.16.8405
14. Fields S, Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989;340(6230):245-246. doi:10.1038/340245a0
15. Johnsson N, Varshavsky A. Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci U S A. 1994;91(22):10340-10344. doi:10.1073/pnas.91.22.10340
16. Andre C, Kim SW, Yu XH, Shanklin J. Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H2O2 to the cosubstrate O2. Proc Natl Acad Sci U S A. 2013;110(8):3191-3196. doi:10.1073/pnas.1218769110
17. Quinn WJ 3rd, et al. Lactate Limits T Cell Proliferation via the NAD(H) Redox State. Cell Rep. 2020;33(11):108500. doi:10.1016/j.celrep.2020.108500
18. San Martín A, Ceballo S, Ruminot I, Lerchundi R, Frommer WB, Barros LF. A genetically encoded FRET lactate sensor and its use to detect the Warburg effect in single cancer cells. PLoS One. 2013;8(2):e57712. doi:10.1371/journal.pone.0057712
19. Nasu Y, Shen Y, Kramer L, Campbell RE. Structure- and mechanism-guided design of single fluorescent protein-based biosensors. Nat Chem Biol. 2021;17(5):509-518. doi:10.1038/s41589-020-00718-x
20. Haas R, et al. Lactate Regulates Metabolic and Pro-inflammatory Circuits in Control of T Cell Migration and Effector Functions. PLoS Biol. 2015;13(7):e1002202. doi:10.1371/journal.pbio.1002202
21. Indra AK, Warot X, Brocard J, Bornert JM, Xiao JH, Chambon P, Metzger D. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res. 1999;27(22):4324-4327. doi:10.1093/nar/27.22.4324

## Table of Contents

* [1. Experimental Aims](#objectives)
* [2. Experimental Text](#experimental-text)
  + [2.1 Design and Results](#sensor-quality)
  + [2.2 Report System](#sensor-assessment)
  + [2.3 Lactate Degradation](#degradation)
* [References](#references)

# LAB Work

Experiment and Results

## Experimental Aims

Our project addresses the lactate-rich tumor microenvironment by engineering a lactate-responsive module that enables immune cells(e.g., CAR-T cells in Car-T therapy) to secrete lactate oxidase (sLOx) and reduce excess lactate. To build this module, a lactate sensing protein is split to two fragments, which are fused to the N-terminal and C-terminal fragments of TEV-protease, respectively. High lactate induces TEV-protease reconstitution, which then cleaves a GV2ER fusion protein to release the transcription factor GV (Gal4-VP64). GV enters the nucleus and activates sLOx expression, lowering lactate concentration and enhancing immune cell anti-tumor efficacy.

## Experimental Text

2.1 Design and Results

2.1.1 Design

Our ultimate goal is to design a highly responsive lactic acid sensor. After an extensive review of the relevant literature, we have formulated the following preliminary plan:

1. Our design is based on the study by Michael et al. In this study, TEV protease is divided into two inactive components, N-TEV and C-TEV. When brought close together through protein-protein interactions (PPIs), they can reassemble into active TEV protease.

2. We are using LIdR as the lactate sensor. When lactate is present in the environment, LIdR undergoes conformational changes. Therefore, we combined the split TEV protease fragments with LIdR fragments. When LIdR senses high levels of lactate, it will trigger the PPI of TEV protease, activating the downstream signaling cascade.

3. We chose GV2ER to respond to TEV activation and mediate the expression of the downstream sLOX gene. GV (Gal4-VP64) is an important transcription factor that is fused to the ERT in the cytoplasm. Between GV and ERT, we inserted a TEV protease cutting site. When the TEV protease is activated, GV can be released from the module and enter the nucleus to activate downstream gene expression.

4. Initially, we chose LOx (lactate oxidase) to target the lactate tumor microenvironment. We inserted a secretion peptide sequence upstream of LOx, enabling its secretion outside the cell as sLOx (secreted lactate oxidase). Additionally, we added the UAS promoter upstream, allowing GV to bind with UAS and activate gene expression. This function is critical in scenarios that require control over lactate levels to maintain microenvironment stability or regulate biological processes.

5. Cell Line Selection: We selected the 293T cell line, which is a commonly used eukaryotic cell line known for easy cultivation and high transfection efficiency. This enables more efficient acquisition of the desired cell phenotype or function when performing genetic manipulations (such as introducing target genes to express the lactate-sensing protein and lactate oxidase). Furthermore, the biological characteristics of 293T cells are well understood, facilitating subsequent experimental research and analysis.

By adhering to this structured plan, we aim to create a responsive lactic acid sensor that can effectively improve the tumor microenvironment and enhance the anti-tumor effects of immune cells.

2.1.2 Results

2.1.2.1 First Construction of the Lactate-Sensing Module

We split the TEV protease into N-terminal and C-terminal segments, which were then connected to the N-terminal and C-terminal of the LIdR protein.（Figure1）

![f1](https://static.igem.wiki/teams/5731/lab/lab1.avif)

Figure 1. This figure illustrates the structural design of the first-generation lactate sensor (LS1.0). It shows that the N-terminal and C-terminal fragments of TEV protease (TEV-N and TEV-C) were respectively connected to the N-terminal and C-terminal of the LldR protein, forming the initial sensor structure for lactate detection.

Therefore, we conducted the first verification experiment. We plated cells in complete medium, transfected the LS1.0 plasmid (constructed based on the LIdR sequence in our components and the pcDNA backbone) into 293T cells, set a lactic acid concentration gradient of 0, 1, and 5 mM for simultaneous stimulation, and then used the dual-luciferase reporter gene for quality detection.

The ideal mechanism of structural change should be as follows.(Figure2)

Figure 2 .This video displays the ideal structural change mechanism of the LS1.0 sensor under lactate stimulation. It visually demonstrates how lactate binding induces conformational changes in the sensor, thereby promoting the reassembly of TEV-N and TEV-C fragments to restore TEV protease activity.

However, the results showed that the TEV enzyme activity was very high even without the addition of lactic acid.(Figure3)

![f3](https://static.igem.wiki/teams/5731/lab/lab3.avif)

Figure 3. This figure presents the experimental results of the LS1.0 sensor verification using the dual-luciferase reporter gene assay. The ordinate (RLU, relative light unit) reflects luciferase enzyme activity; the results show that even without lactate stimulation (0mM), the TEV enzyme activity of the LS1.0 group is very high, indicating background activity issues in the first-generation sensor.

After team discussion, we speculate that this was because the split TEV enzyme was too close to each other on the LIdR protein, causing the TEV to become active and function even before lactic acid binding. Therefore, we improved it to develop LS2.0 - the second-generation lactate sensor.

Elevated lactic acid levels did not significantly alter enzyme production.

2.1.2.2 Second Construction of the Lactate-Sensing Module

Based on the failure of the previous generation sensor, we made the first improvement to the original sensor: we split the LIdR protein into two parts (N-terminal and C-terminal) and connected them to the N-terminal and C-terminal of the TEV protease, respectively, completing the design of a brand-new sensor.(Figure4)

Figure 4. This figure shows the structural design of the second-generation lactate sensor (LS2.0). Unlike LS1.0, the LldR protein was split into N-terminal (Lac-N) and C-terminal (Lac-C) fragments, which were then respectively connected to TEV-N and TEV-C to form a new sensor structure, aiming to reduce background TEV activity.

Subsequently, we conducted the second experimental verification, and the results are as follows.(Figure5)

![f5](https://static.igem.wiki/teams/5731/lab/lab5.avif)

Figure5. This figure presents the experimental results of the LS2.0 sensor verification using the dual-luciferase reporter gene assay. The ordinate (RLU) represents TEV enzyme activity; similar to LS1.0, the results indicate that the LS2.0 group still exhibits high TEV enzyme activity in the absence of lactate (0mM), meaning the background activity problem was not resolved.

However, during the experiment, we found that the TEV enzyme activity was still high without the addition of lactic acid. Therefore, we decided to further improve our sensor based on the above.

2.1.2.3 Third Construction of the Lactate-Sensing Module

In this improvement, we changed the connection mode of the N-terminal and C-terminal of the two proteins and constructed 8 combination modes.(Figure6)(Figure7)(Figure8)

![f6](https://static.igem.wiki/teams/5731/lab/lab6.avif)

Figure 6.This figure schematically shows the two connection modes of LldR fragments (Lac-N, Lac-C) and TEV fragments (TEV-N, TEV-C) before optimizing the LS3.x series sensors. It contrasts the original fixed connection mode (Lac-N-TEV-N and Lac-C-TEV-C) with the subsequent diversified combination design ideas.

Figure 7.This figure details the 8 different combination modes of LldR fragments (Lac-N, Lac-C) and TEV fragments (TEV-N, TEV-C) for constructing the LS3.1-LS3.8 sensors. Each combination represents a unique fusion sequence of the sensing module and TEV enzyme fragments.

Instead, we changed the connection mode of the N-terminal and C-terminal of the two proteins and constructed 8 combination modes.(Figure8)

![f8](https://static.igem.wiki/teams/5731/lab/f8.webp)

Figure 8.This figure provides a visual diagram of the structural composition of each sensor in the LS3.1-LS3.8 series. It clearly marks the connection relationship between Lac-N/Lac-C and TEV-N/TEV-C in each sensor, serving as a design basis for subsequent background screening.

First, we conducted a background screening to investigate the response degree of each group of sensors without the addition of lactic acid. The screening results are shown in the figure.(Figure9)

![f9](https://static.igem.wiki/teams/5731/lab/lab9.avif)

Figure 9. This figure presents the background screening results of the LS3.1-LS3.8 sensors. The ordinate (RLU) reflects the TEV enzyme activity of each sensor without lactate stimulation; the results help screen out sensors with low background activity (e.g., LS3.5, LS3.6, LS3.8).

Finally, we screened out three combinations with low background, namely LS3.5, LS3.6, and LS3.8. We finally demonstrate that LS3.5 is the sensor we need with high sensitivity and stability. Our modeling results also corroborate this.Our modeling results also confirm this. For details, please visit our [Model page](https://2025.igem.wiki/csu-china/model).The results are shown in the figure.(Figure10)

![f10](https://static.igem.wiki/teams/5731/lab/lab10.avif)

Figure 10 displays the sensitivity and stability verification results of the low-background sensors that were screened. Ls3.5 exhibits the lowest background, and its signal increases with higher lactate input, whereas Ls3.6 and Ls3.8 do not show this trend.

2.2 Report System

2.2.1 GV2ER

2.2.1.1 The Structure of GV2ER

To accurately detect the functional efficacy and stability of the lactate sensor within cells, we constructed the GV2ER reporter gene plasmid.(Figure 11)

![f11](https://static.igem.wiki/teams/5731/lab/lab11.avif)

Figure 11. GV2ER:The "G" stands for the yeast Gal4 DNA-binding domain. In transcriptional regulation, this domain can specifically recognize and bind to DNA sequences containing Gal4 response elements.The "V" represents the herpes simplex virus VP16 transactivation domain, which possesses a potent function of activating transcription. When fused with the Gal4 DNA-binding domain to form "GV", once GV binds to the corresponding DNA sequence, VP16 can recruit transcription-related enzymes and protein factors, promote the initiation and progression of transcription, and thereby trigger the expression of downstream reporter genes (such as the firefly luciferase gene G5-Luci or the enhanced green fluorescent protein gene G5-EGFP).The "2ER" denotes two copies of the estrogen receptor mutant ERT2. The ERT2 domain is a mutated estrogen receptor domain that is sensitive to 4-hydroxy-tamoxifen (4-OHT) but insensitive to estrogen. In the absence of 4-OHT in cells, ERT2 traps the proteins linked to it in the cytoplasm. In GV-2ER, the two ERT2 domains are located at the N-terminus and C-terminus of GV, respectively. Acting like "molecular locks", they firmly sequester the GV transcription factor in the cytoplasm, preventing it from entering the nucleus to initiate transcription. Only after the ERT2 domains are removed via cleavage by TEV protease can GV enter the nucleus and exert its transcriptional activation function.

The lactate sensor, as a key protein for sensing changes in lactate concentration inside and outside cells, undergoes conformational changes or mediates protein-protein interactions (PPI) when bound to its ligand (lactate); these molecular events are crucial links in regulating physiological processes such as cellular metabolism and signal transduction.

By specifically capturing the molecular interaction events related to the lactate sensor through the GV2ER system, the response efficiency (effect) of the sensor to lactate and its functional maintenance capability (stability) under different time and environmental conditions can be quantitatively evaluated. This provides a reliable detection method for in-depth analysis of the physiological functions of the lactate sensor and the mechanisms of related diseases.

2.2.1.2 The Mechanism of Action of GV2ER

The entire system enables the detection of target protein-protein interactions (PPI) through a molecular cascade process: "TEV protease fragment complementation and activation → release of GV transcription factor → transcriptional activation in the nucleus → reporter gene expression".(Figure 12)

![f12](https://static.igem.wiki/teams/5731/lab/lab12.avif)

Figure 12. In this process, lactate enters the cell. Lac-N and Lac-C, along with TEV-N and TEV-C, bind to form a complex. This complex cleaves the ERT domains from GV via TEV protease activity. Freed GV then enters the nucleus, binds to the 5×UAS sequence, and initiates the expression of the downstream reporter gene sLOx.

In the absence of target protein-protein interactions (PPI), the system remains in an initial quiescent state. The GV transcription factor, flanked by TEV cleavage sites (tevS) and linked to ERT2, is "dragged" and anchored in the cytoplasm by ERT2, thereby being unable to translocate into the nucleus through nuclear pores. Consequently, downstream reporter genes (e.g.G5-Luci, G5-EGFP) remain unexpressed due to the lack of transcriptional activation signals.

When the target protein-protein interaction (PPI) occurs, the system is triggered and activated. The two proteins involved in the target PPI are fused with the inactive N-terminal TEV fragment (N-TEV) and C-terminal TEV fragment (C-TEV), respectively. When these two target proteins interact, the fused N-TEV and C-TEV fragments undergo reconstitution due to the proximity of the proteins, forming a fully active TEV protease. Serving as the signal sensing module of the entire system, this step converts the molecular event of PPI occurrence into a biochemical signal of TEV protease activation.

Subsequently, the process proceeds to a critical step: upon the formation of the active TEV protease, it specifically recognizes and binds to the tevS site located between the GV transcription factor and ERT2. Through proteolytic hydrolysis, the TEV protease cleaves the peptide bond at the tevS site, leading to the dissociation of the GV transcription factor from the ERT2 domain. At this point, the GV transcription factor, freed from the constraint of ERT2, is no longer anchored by heat shock proteins (HSPs) and gains the ability to translocate into the nucleus. With this, the system completes signal transduction and shifts from a quiescent state to an activation-ready state.

The released GV transcription factor translocates into the nucleus through nuclear pores, initiates a transcriptional cascade, and generates detectable reporter signals (e.g., fluorescence, chemiluminescence).

The signal intensity generated by reporter gene expression is positively correlated with the occurrence efficiency of the target PPI. By detecting fluorescence intensity (EGFP) or chemiluminescence intensity (Luci), the occurrence frequency and strength of the target PPI can be indirectly quantified, enabling visual and quantitative analysis of PPI.

2.2.2 Fluorescence Detection

In the lactate sensor system of this study, a dual-luciferase reporter gene was employed for quality assessment. The dual-luciferase reporter gene takes Renilla Luciferase and Firefly Luciferase as its core components.

2.2.2.1 The Structure of pGL4.35

Among them, Firefly Luciferase serves as the "functional reporter signal", and we designed the plasmid pGL4.35 [luc2P/9XGAL4UAS/Hygro] (provided by Promega).(Figure 13)

![f13](https://static.igem.wiki/teams/5731/lab/lab13.avif)

Figure 13. This figure presents the plasmid map of pGL4.35 [luc2P/9×GAL4UAS/Hygro] (provided by Promega), a core plasmid for dual-luciferase reporter gene detection in the experiment. It clearly marks multiple key functional elements, including the Ad promoter (driving gene transcription), 9×GAL4 UAS (binding to GAL4 protein to regulate gene expression), luciferase gene (serving as a reporter gene to produce detectable fluorescence), hPEST sequence (regulating luciferase stability), SV40 ori (facilitating plasmid replication in specific cells), HygR (hygromycin resistance gene for cell screening), AmpR (ampicillin resistance gene for prokaryotic system screening), and poly(A) signal (ensuring correct processing of transcription products). These elements work synergistically to enable the plasmid to be used for detecting the transcriptional activation efficiency mediated by the GV transcription factor.

The **pGL4.35 [luc2P/9XGAL4UAS/Hygro] plasmid** contains multiple key elements:The Ad promoter (adenovirus promoter) drives the transcription of relevant genes;

The **9X GAL4 UAS** (9 copies of GAL4 upstream activation sequence) can bind to the GAL4 protein to regulate gene expression;

**Luciferase** (luciferase gene) acts as a reporter gene, which produces fluorescence for detection after expression;

The **hPEST sequence** regulates the stability of luciferase;

The **SV40 ori** (SV40 origin of replication) facilitates plasmid replication in specific cells, while the SV40 promoter drives the transcription of downstream genes;

The **HygR** (hygromycin resistance gene) confers hygromycin resistance to cells, enabling the selection of cells containing the plasmid;

The **AmpR** (ampicillin resistance gene) allows selection in prokaryotic systems;

The **poly (A) signal** (polyadenylation signal) ensures the correct processing of transcription products;

Additional elements such as the signal pause site are also included.

All these elements work synergistically, allowing the plasmid to be used in studies related to gene expression regulation and other relevant research areas.

#### The Mechanism of Action of Luciferase

The expression or activity of luciferase (luciferase gene) directly depends on the detection of protein-protein interactions (PPI) by the sensor. In the GV2ER system, when the target PPI occurs—inducing the reconstitution and reactivation of TEV protease fragments—the active TEV protease cleaves the membrane-anchored or cytoplasm-retained transcription factor. The released GV then translocates into the nucleus and drives the expression of the downstream Firefly luciferase gene (G5-Luci).

2.3 Lactate Degradation

![f14](https://static.igem.wiki/teams/5731/lab/lab14.avif)

Figure 14 . This figure shows the structural design of the engineered secreted lactate oxidase (sLOx). It illustrates that a signal sequence derived from the rat follicle-stimulating hormone β-subunit (rat FSHB) was fused to the N-terminus of the lactate oxidase (LOx) sequence, and a FLAG tag was also included in the structure (with the total length marked as approximately 1250 base pairs). The rat FSHB signal sequence is responsible for directing the newly synthesized sLOx protein into the cell secretory pathway, ensuring that sLOx is secreted extracellularly to exert its lactate-degrading function, rather than being retained in the cytoplasm.

Subsequently, to ensure the smooth implementation of the lactic acid detection assay—aimed at verifying the performance and stability of the sensors we had screened—we replaced the Luciferase gene (which had served as a reporter gene in the previous sensor screening experiments) with the optimized sLOx gene. Based on this modification, we designed an entirely new plasmid. (Figure 15)

![f15](https://static.igem.wiki/teams/5731/lab/lab15.avif)

Figure 15. This figure presents the map of the novel plasmid designed for verifying sLOx function. Based on the original plasmid used in sensor screening (which contained the Luciferase reporter gene), the Luciferase gene was replaced with the optimized sLOx gene. The plasmid retains key elements required for gene expression and cell screening, enabling the expression of sLOx to be regulated by the upstream lactate sensor system and facilitating subsequent verification of sLOx secretion and lactate-degrading activity.

We co-transformed the sLOx plasmid and GV2ER into HEK293 T cells, cultured them in medium containing five mmol lactic acid, and then extracted cell lysates and culture medium separately after 24 hours. Expression of LOx in both cell lysates and culture medium was detected using the flac antibody (located at the loxC site). As shown in the figure, lox can be induced by lactic acid and secreted into the culture medium.

![f16](https://static.igem.wiki/teams/5731/lab/lab16.avif)

Figure 16. This figure displays the results of the Western blot assay used to verify the secretion of sLOx protein. The blot includes two sample types: culture medium (extracellular fluid) and cell lysate (intracellular fluid), with both NC (negative control) and LS3.5 (sensor-sLOx system) groups tested. The presence of the sLOx protein band in the culture medium of the LS3.5 group (while no obvious band is detected in the NC group’s culture medium) confirms that the engineered sLOx protein was successfully secreted from 293T cells into the extracellular space.

Further results show that the expression of sLOx can significantly reduce extracellular lactate concentration. Figure 17 shows the results of our lactate degradation experiment:

![f17](https://static.igem.wiki/teams/5731/lab/lab17.avif)

Figure 17. This figure shows the results of the extracellular lactate degradation assay. The ordinate represents the relative concentration of extracellular lactate, and the abscissa shows the NC group and the LS3.5 group (sensor-sLOx system group). The results clearly indicate that compared with the NC group, the extracellular lactate concentration in the LS3.5 group is significantly lower, directly demonstrating that the secreted sLOx protein has a stable and efficient lactate-degrading function, which can reduce the local extracellular lactate concentration as expected.

## References

1. Wintgens JP, et al. Monitoring activities of receptor tyrosine kinases using a universal adapter in genetically encoded split TEV assays. Cellular and Molecular Life Sciences. 2019;76:1185-1199. doi:10.1007/s00018-018-03003-2
2. Li X, et al. Ultrasensitive sensors reveal the spatiotemporal landscape of lactate metabolism in physiology and disease. Cell Metabolism. 2023;35:200-211.e9. doi:10.1016/j.cmet.2022.10.002
3. Markert CL. Cell Biochem Funct. 1984;2(3):131-134.
4. Feng Y, et al. Cancer Med. 2018;7(12):6124-6136.
5. Cristescu ME, Innes DJ, Stillman JH, Crease TJ. BMC Evol Biol. 2008;8:268.
6. Forkasiewicz A, et al. Cell Mol Biol Lett. 2020;25:35.
7. Claps G, et al. Nat Rev Clin Oncol. 2022;19(12):749-762.
8. Aguilera L, Campos E, Giménez R, Badía J, Aguilar J, Baldoma L. Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. J Bacteriol. 2008;190(8):2997-3005. doi:10.1128/JB.02013-07
9. Faubert B, et al. Lactate Metabolism in Human Lung Tumors. Cell. 2017;171(2):358-371.e9. doi:10.1016/j.cell.2017.09.019
10. Kapust RB, Waugh DS. Controlled intracellular processing of fusion proteins by TEV protease. Protein Expr Purif. 2000;19(2):312-318. doi:10.1006/prep.2000.1251
11. Meister SW, et al. Engineering of TEV protease variants with redesigned substrate specificity. Biotechnol J. 2023;18:e2200625. doi:10.1002/biot.202200625
12. Wehrman T, Kleaveland B, Her JH, Balint RF, Blau HM. Protein-protein interactions monitored in mammalian cells via complementation of beta -lactamase enzyme fragments. Proc Natl Acad Sci U S A. 2002;99(6):3469-3474. doi:10.1073/pnas.062043699
13. Rossi F, Charlton CA, Blau HM. Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc Natl Acad Sci U S A. 1997;94(16):8405-8410. doi:10.1073/pnas.94.16.8405
14. Fields S, Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989;340(6230):245-246. doi:10.1038/340245a0
15. Johnsson N, Varshavsky A. Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci U S A. 1994;91(22):10340-10344. doi:10.1073/pnas.91.22.10340
16. Andre C, Kim SW, Yu XH, Shanklin J. Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H2O2 to the cosubstrate O2. Proc Natl Acad Sci U S A. 2013;110(8):3191-3196. doi:10.1073/pnas.1218769110
17. Quinn WJ 3rd, et al. Lactate Limits T Cell Proliferation via the NAD(H) Redox State. Cell Rep. 2020;33(11):108500. doi:10.1016/j.celrep.2020.108500
18. San Martín A, Ceballo S, Ruminot I, Lerchundi R, Frommer WB, Barros LF. A genetically encoded FRET lactate sensor and its use to detect the Warburg effect in single cancer cells. PLoS One. 2013;8(2):e57712. doi:10.1371/journal.pone.0057712
19. Nasu Y, Shen Y, Kramer L, Campbell RE. Structure- and mechanism-guided design of single fluorescent protein-based biosensors. Nat Chem Biol. 2021;17(5):509-518. doi:10.1038/s41589-020-00718-x
20. Haas R, et al. Lactate Regulates Metabolic and Pro-inflammatory Circuits in Control of T Cell Migration and Effector Functions. PLoS Biol. 2015;13(7):e1002202. doi:10.1371/journal.pbio.1002202
21. Indra AK, Warot X, Brocard J, Bornert JM, Xiao JH, Chambon P, Metzger D. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res. 1999;27(22):4324-4327. doi:10.1093/nar/27.22.4324