iGEM 2021 — Team British_Columbia

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:British Columbia/Notebook

UBC iGEM 2021

[![detecTME logo](https://static.igem.org/mediawiki/2021/1/18/T--British_Columbia--detecTME_logo_light_no_text.svg)](https://2021.igem.org/Team%3ABritish_Columbia)

* Project
  + [Description](https://2021.igem.org/Team%3ABritish_Columbia/Description)
  + [Design](https://2021.igem.org/Team%3ABritish_Columbia/Design)
  + [Implementation](https://2021.igem.org/Team%3ABritish_Columbia/Implementation)
  + [Partnerships](https://2021.igem.org/Team%3ABritish_Columbia/Partnership)
* Experiments
  + [Experimental Design](https://2021.igem.org/Team%3ABritish_Columbia/Experiments)
  + [Engineering Success](https://2021.igem.org/Team%3ABritish_Columbia/Engineering)
  + [Results](https://2021.igem.org/Team%3ABritish_Columbia/Results)
  + [Parts](https://2021.igem.org/Team%3ABritish_Columbia/Parts)
  + [Contributions](https://2021.igem.org/Team%3ABritish_Columbia/Contribution)
  + [Protocols](https://2021.igem.org/Team%3ABritish_Columbia/Protocols)
  + [Lab Notebook](https://2021.igem.org/Team%3ABritish_Columbia/Notebook)
  + [Safety](https://2021.igem.org/Team%3ABritish_Columbia/Safety)
* Models
  + [Mathematical Modelling](https://2021.igem.org/Team%3ABritish_Columbia/Model)
  + [Software Simulations](https://2021.igem.org/Team%3ABritish_Columbia/Model)
* Human Practices
  + [Integrated HP](https://2021.igem.org/Team%3ABritish_Columbia/Human_Practices)
  + [Outreach](https://2021.igem.org/Team%3ABritish_Columbia/Communication)
  + [Education](https://2021.igem.org/Team%3ABritish_Columbia/Education)
  + [Collaborations](https://2021.igem.org/Team%3ABritish_Columbia/Collaborations)
  + [Book Chapter](https://2021.igem.org/Team%3ABritish_Columbia/Book_Chapter)
* Team
  + [Team Members](https://2021.igem.org/Team%3ABritish_Columbia/Team)
  + [Attributions](https://2021.igem.org/Team%3ABritish_Columbia/Attributions)
  + [Sponsors](https://2021.igem.org/Team%3ABritish_Columbia/Sponsors)
* [Achievements](https://2021.igem.org/Team%3ABritish_Columbia/Achievements)

EXPERIMENTS

# Lab Notebook

Below you can find our laboratory notebook for the months wet-lab was active.

All protocols used in this notebook, unless otherwise specified, can be found on our [Protocols](https://2021.igem.org/Team%3ABritish_Columbia/Protocols) page.

[![](https://static.igem.org/mediawiki/2021/f/f7/T--British_Columbia--UBC_iGEM_white_logo.png)](https://www.ubcigem.com/)

###### Project

[Description](https://2021.igem.org/Team%3ABritish_Columbia/Description)

[Design](https://2021.igem.org/Team%3ABritish_Columbia/Design)

[Implementation](https://2021.igem.org/Team%3ABritish_Columbia/Implementation)

[Partnerships](https://2021.igem.org/Team%3ABritish_Columbia/Partnership)

###### Experiments

[Experiments](https://2021.igem.org/Team%3ABritish_Columbia/Experiments)

[Engineering](https://2021.igem.org/Team%3ABritish_Columbia/Engineering)

[Results](https://2021.igem.org/Team%3ABritish_Columbia/Results)

[Parts](https://2021.igem.org/Team%3ABritish_Columbia/Parts)

[Contributions](https://2021.igem.org/Team%3ABritish_Columbia/Contribution)

[Protocols](https://2021.igem.org/Team%3ABritish_Columbia/Protocols)

[Lab Notebook](https://2021.igem.org/Team%3ABritish_Columbia/Notebook)

[Safety](https://2021.igem.org/Team%3ABritish_Columbia/Safety)

###### Models

[Mathematical Modelling](https://2021.igem.org/Team%3ABritish_Columbia/Model)

[Software Simulations](https://2021.igem.org/Team%3ABritish_Columbia/Model)

###### Human Practices

[Integrated HP](https://2021.igem.org/Team%3ABritish_Columbia/Human_Practices)

[Outreach](https://2021.igem.org/Team%3ABritish_Columbia/Communication)

[Education](https://2021.igem.org/Team%3ABritish_Columbia/Education)

[Collaborations](https://2021.igem.org/Team%3ABritish_Columbia/Collaborations)

[Book Chapter](https://2021.igem.org/Team%3ABritish_Columbia/Book_Chapter)

###### Team

[Members](https://2021.igem.org/Team%3ABritish_Columbia/Team)

[Attributions](https://2021.igem.org/Team%3ABritish_Columbia/Attributions)

[Sponsors](https://2021.igem.org/Team%3ABritish_Columbia/Sponsors)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:British Columbia/Engineering

UBC iGEM 2021

[![detecTME logo](https://static.igem.org/mediawiki/2021/1/18/T--British_Columbia--detecTME_logo_light_no_text.svg)](https://2021.igem.org/Team%3ABritish_Columbia)

* Project
  + [Description](https://2021.igem.org/Team%3ABritish_Columbia/Description)
  + [Design](https://2021.igem.org/Team%3ABritish_Columbia/Design)
  + [Implementation](https://2021.igem.org/Team%3ABritish_Columbia/Implementation)
  + [Partnerships](https://2021.igem.org/Team%3ABritish_Columbia/Partnership)
* Experiments
  + [Experimental Design](https://2021.igem.org/Team%3ABritish_Columbia/Experiments)
  + [Engineering Success](https://2021.igem.org/Team%3ABritish_Columbia/Engineering)
  + [Results](https://2021.igem.org/Team%3ABritish_Columbia/Results)
  + [Parts](https://2021.igem.org/Team%3ABritish_Columbia/Parts)
  + [Contributions](https://2021.igem.org/Team%3ABritish_Columbia/Contribution)
  + [Protocols](https://2021.igem.org/Team%3ABritish_Columbia/Protocols)
  + [Lab Notebook](https://2021.igem.org/Team%3ABritish_Columbia/Notebook)
  + [Safety](https://2021.igem.org/Team%3ABritish_Columbia/Safety)
* Models
  + [Mathematical Modelling](https://2021.igem.org/Team%3ABritish_Columbia/Model)
  + [Software Simulations](https://2021.igem.org/Team%3ABritish_Columbia/Model)
* Human Practices
  + [Integrated HP](https://2021.igem.org/Team%3ABritish_Columbia/Human_Practices)
  + [Outreach](https://2021.igem.org/Team%3ABritish_Columbia/Communication)
  + [Education](https://2021.igem.org/Team%3ABritish_Columbia/Education)
  + [Collaborations](https://2021.igem.org/Team%3ABritish_Columbia/Collaborations)
  + [Book Chapter](https://2021.igem.org/Team%3ABritish_Columbia/Book_Chapter)
* Team
  + [Team Members](https://2021.igem.org/Team%3ABritish_Columbia/Team)
  + [Attributions](https://2021.igem.org/Team%3ABritish_Columbia/Attributions)
  + [Sponsors](https://2021.igem.org/Team%3ABritish_Columbia/Sponsors)
* [Achievements](https://2021.igem.org/Team%3ABritish_Columbia/Achievements)

EXPERIMENTS

# Engineering Success

[Introduction](#Intro)

---

[Genetic Circuits](#Circuits)

---

[Lactate Induction Experiments](#LactateInduction)

## **INTRODUCTION**

---

In harnessing genetic and biological engineering for our overall project goals, we undergo smaller iterations of the engineering design process. From building and testing circuits, to assays, to engineered organisms, we learn from, and consequently iterate, each design until the optimal one is reached. Below we describe  two different cycles we went through in our wet-lab, where we learned from our own designs and tweaked them accordingly to reach more desired results.

## **GENETIC CIRCUITS**

---

The iterations of our genetic circuit’s design  differed in the way they implemented the AND-gate system to ensure more sensitive readout of immune activity.

## **Design Iteration #1**

While the biomarkers and hence the inducible promoters were the same as the current workflow, with our initial design, the activation of the sicA promoter resulted in production of a lactate permease to transport lactate into the cell, which would then activate the lactate-inducible promoter. In turn, the activation of this promoter would express luciferase with a secretion tag, producing our reporter for secretion in urine.

Steps:

**1)** Produce a stable line of bacteria that have their endogenous lactate transporters knocked out by CRISPR

**2)** TNFa induces the activation of the sicA promoter which allows for the prosecution of lactate transporters

**3)** If lactate is present in the extracellular environment, it enters the cell through the transporters and activates the lactate-inducible promoter, leading to the production of nanoluciferase with a Salmonella secretion tag

**4)** The nanoluciferase enters the bloodstream and is detected in the urine

## **Circuit Build #1**

![Circuit #1](https://static.igem.org/mediawiki/2021/9/94/T--British_Columbia--UBC_old_circuit.png)

## **Conceptual Test #1**

After designing this circuit and seeking feedback from our graduate advisors, we uncovered various fundamental issues with the setup and logic:

* Salmonella  naturally takes up lactate as part of its metabolism(Gillis et. al, 2019), which acts as an integral nutrient source. Hence, each cell must already have a way of transporting lactate through its cell wall, likely through theexistence of lactate permease in its membrane. Knocking out lactate permease, as was proposed in our original design, would therefore interrupt Salmonella metabolism and possibly interrupt normal function of our chassis, or even be lethal to the cell.

* Luciferase secretion by the secretion tag we designed would release the protein into a host cell rather than the extracellular matrix.

## **Learn #1**

The solution to this concern would be to avoid knocking out lactate permease. However, endogenous transport of lactate into the cell abolishes the AND-gate concept in our system since only lactate would be required to produce the reporter. Since lactate is already naturally being uptaken by the cell, this results in the constant production of our reporter in lactate-rich environments, even without the presence of our second biomarker. As explained in our [Design](https://2021.igem.org/Team%3ABritish_Columbia/Design) page, given that lactate on its own is not a strong enough indication of tumour presence, our system would not be a very reliable tool for characterizing immune activity in tumours without the AND-gate.

## **Design Iteration #2**

Since these issues are all caused by making lactate permease mediate the AND-gate, we decided to produce a system that would not require this transport protein but keep the AND-gate. It would instead follow the logic below:

**1)** Produce a wild-type Salmonella cell line that expresses both constructs with TNFa-inducible promoter and the lactate-inducible promoter.

**2)** The construct with the TNFa-inducible promoter would drive expression of components luxA, luxB, and luxC of the lux operon, and the lactate-inducible promoter would drive expression of components luxC, luxD, and luxE of the lux operon.

**3)** Since luciferase is produced by luxA and luxB, only when both TNFa and lactate are present will all components of luciferin be produced. Split-protein systems rely on hydrophobic forces automatically bringing both halves back together. Since hydrophobic residues are usually protected within the interior of the protein, but become exposed when split, it becomes energetically favorable to rejoin the split halves of the protein to prevent hydrophobic interactions with polar molecules. Hence, once all components of luciferin are produced, the full protein will assemble and luminesce, producing the reporter only in the presence of both biomarkers.

![Circuit #2](https://static.igem.org/mediawiki/2021/6/68/T--British_Columbia--UBC_engineering_new_circuit.png)

In conclusion, the genetic circuits we constructed, which were the central base for wet-lab efforts, went through a redesign that stemmed from careful consideration by undergraduate and graduate advisors.

## **LACTATE INDUCTION EXPERIMENTS**

---

As the lactate-inducible promoter gets induced once lactate binds to its substrate, we measured the promoter's activity by testing different concentrations of lactate with a plate reader. On each run of our induction experiment, we learned from the previous trial run and built upon its design to reach the ideal experimental setup.

## **Design and Build**

We designed the experiment to be performed on a Varioskan plate reader and a 96 well plate.

Multiple different strains of cells were used to ensure the integrity of our experiment. Our construct of interest (pLactate + lldR + RFP plasmid) was transformed into chemically competent E. coli and screened with chloramphenicol. The construct was built as described in the circuit construction section of our Experimental design page. As a positive control, we used a constitutively expressing RFP cell strain to determine the ceiling of possible fluorescence from our lactate regulated construct. For a baseline/negative control, we used an E. coli cell strain with  an “empty” vector (contains a pSB1C3 plasmid without an insert). Another negative control for the plate was a lane of “only media”. The cells were to be seeded into the wells at a cell density of 0.1 OD600. The cell concentration would allow us the ability to see the growth of the cells while shortening the time it took for the cells to reach the stationary phase

We chose specific lactate concentrations for the media we were using. These concentrations were to elucidate the viability of our construct in identifying healthy tissue vs tumorous tissues. The values were determined through lactate measurements in literature. We settled on 0mM, 2mM, 5mM, 15mM, 40mM and 100mM lactate since these values gave us good enough separation to determine the viability of our construct. Since the plate and plate reader worked best at a volume/well of 200-300 uL, we decided on a total volume of 250 uL (200 uL media, 50 uL cells). The media would be plated first, and then the cells added. Concentrations would be calculated to take into account the lactate dilution factor that would happen after the cells were added to the media (e.g instead of plating LB media with 100 mM lactate, 125 mM would be made instead). Similarly, this dilution factor would be taken into consideration for cell concentrations. Both of this would allow us to keep the concentration of lactate and cells to be properly controlled.

## **Test**

On the day before the experiment was run, we inoculated RFP cells, cells with an empty plasmid (e.g. plasmid pSCB1C3 without an insert), and the cells with the construct (lactate inducible promoter system and reporter protein gene). Then, we made and diluted the lactate solution to the appropriate concentrations to be put in the reader.

On the day of the experiment, we first prepped the inoculations. Using the spectrometer, we  diluted the cells to an OD600 of the cells to 0.5 OD600. Then, we loaded our media and our cells into a 96 plate using a multichannel pipettor, and ran the reading of the plate. The plate reader was run for 18 hours overnight and the data was collected the following morning.

## **Learn and Redesign**

Upon discussing results with our advisors, we added a few improvements for our second lactate induction experiment run. First, we made sure to wash the cells in LB before employing them in our experiment. Washing the cells enables us to remove certain metabolites that may have accumulated overnight and prevent them from interfering with our cells. This was done through pelleting down the cells and resuspending it with fresh LB (repeated twice). Second, we ensured to add an OD read in our plate reader cycles so that the fluorescence reads could be normalized to the cell growth/population. Third, we switched out our clear, white plates for clear, black plates for more accurate fluorescence readings.

In this iteration, the OD600 reads allowed us to normalize the fluorescence reads. After analysis, this returned a more expected pattern where fluorescence increased with lactate concentrations, proving that our construct does work. Early on however, bubbles at the start (0 h timepoint) skewed our initial reads, which is something we need to take into account next time.

## **Build #2**

A large part of our proof-of-concept wet-lab work would be to corroborate that our construct works in the chassis (S. typhimurium) of our choosing. With this in mind, we transformed our construct, along with a RFP-containing plasmid and an "empty" vector, into electrocompetent Salmonella cells. We then ran a similar induction experiment to those outlined above. Learning even more from our second experiment run, we now understood that bubbles produced from pipetting introduce early experimental artifacts. We also took on additional lactate concentrations (0. 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 40, 100 mM lactate) and decreased the times between our runs (1 hour to 10 mins) to increase the overall resolution of our data from the experiment.

![Growth Curve from Trial 2 Data](https://static.igem.org/mediawiki/2021/f/f3/T--British_Columbia--GrowthCurve_Trial2.png)

Growth Curve (Trial 2)

![Normalized Fluorescence vs. Lactate Concentration](https://static.igem.org/mediawiki/2021/8/8d/T--British_Columbia--LactateInduction_Trial2.png)

Normalized Fluorescence vs. Lactate Concentration (Trial 2)

## **Build #3**

A large part of our proof of concept wet lab work would be to corroborate that our construct works in the chassis (S. typhimurium) of our choosing. With this in mind, we transformed our construct, along with a RFP containing plasmid and an “empty” vector, into electrocompetent Salmonella cells. We then ran a similar induction experiment from the ones outlined above. Learning even more from our second experiment run, we now understood that bubbles produced from pipetting introduces early experimental artifacts. Taking this into account, we made sure to start incubating and reading after the bubbles disappeared. We also took on additional lactate concentrations (0. 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 40, 100 mM lactate) and decreased the times between our runs (1 hour to 10 mins) to increase the overall resolution of our data from the experiment.

[**GO TO RESULTS**](https://2021.igem.org/Team%3ABritish_Columbia/Results)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:British Columbia/Experiments

UBC iGEM 2021

[![detecTME logo](https://static.igem.org/mediawiki/2021/1/18/T--British_Columbia--detecTME_logo_light_no_text.svg)](https://2021.igem.org/Team%3ABritish_Columbia)

* Project
  + [Description](https://2021.igem.org/Team%3ABritish_Columbia/Description)
  + [Design](https://2021.igem.org/Team%3ABritish_Columbia/Design)
  + [Implementation](https://2021.igem.org/Team%3ABritish_Columbia/Implementation)
  + [Partnerships](https://2021.igem.org/Team%3ABritish_Columbia/Partnership)
* Experiments
  + [Experimental Design](https://2021.igem.org/Team%3ABritish_Columbia/Experiments)
  + [Engineering Success](https://2021.igem.org/Team%3ABritish_Columbia/Engineering)
  + [Results](https://2021.igem.org/Team%3ABritish_Columbia/Results)
  + [Parts](https://2021.igem.org/Team%3ABritish_Columbia/Parts)
  + [Contributions](https://2021.igem.org/Team%3ABritish_Columbia/Contribution)
  + [Protocols](https://2021.igem.org/Team%3ABritish_Columbia/Protocols)
  + [Lab Notebook](https://2021.igem.org/Team%3ABritish_Columbia/Notebook)
  + [Safety](https://2021.igem.org/Team%3ABritish_Columbia/Safety)
* Models
  + [Mathematical Modelling](https://2021.igem.org/Team%3ABritish_Columbia/Model)
  + [Software Simulations](https://2021.igem.org/Team%3ABritish_Columbia/Model)
* Human Practices
  + [Integrated HP](https://2021.igem.org/Team%3ABritish_Columbia/Human_Practices)
  + [Outreach](https://2021.igem.org/Team%3ABritish_Columbia/Communication)
  + [Education](https://2021.igem.org/Team%3ABritish_Columbia/Education)
  + [Collaborations](https://2021.igem.org/Team%3ABritish_Columbia/Collaborations)
  + [Book Chapter](https://2021.igem.org/Team%3ABritish_Columbia/Book_Chapter)
* Team
  + [Team Members](https://2021.igem.org/Team%3ABritish_Columbia/Team)
  + [Attributions](https://2021.igem.org/Team%3ABritish_Columbia/Attributions)
  + [Sponsors](https://2021.igem.org/Team%3ABritish_Columbia/Sponsors)
* [Achievements](https://2021.igem.org/Team%3ABritish_Columbia/Achievements)

EXPERIMENTS

# Experimental Design

[Introduction](#Intro)

---

[Circuit Construction](#CircuitConstruction)

---

[Circuit Validation](#CircuitValidation)

---

[Inducible Promoter Discovery](#PromoterDiscovery)

## **INTRODUCTION**

---

Based on our [Project Design](https://2021.igem.org/Team%3ABritish_Columbia/Design) decisions, we set out to experimentally validate our project’s four main components:

* TNFa-induction
* Lactate-induction
* Split-lux operon AND-gate system
* Inducible promoter screening

## **CIRCUIT CONSTRUCTION**

---

### **Construction of psicA Characterization Circuit**

The sicA promoter regulates the expression of secreted effort proteins such as  Salmonella Invasion Protein (Sip) A in  Salmonella (Temme et al, 2008). Ma et al (2010) reported that upon the induction of human TNFa, there was a significant increase of SipA protein production. Hence we hypothesized that sicA was involved in a regulatory pathway in Salmonella that was responsive to TNFa. Since TNFa was identified as a potential immune marker of interest in tumour microenvironments, we decided to further characterize its inductive function to the psicA promoter as a proof-of-concept. Following this proof-of-concept, this validated inducible system would be used to produce a reporter in the presence of both TNFa and our second biomarker, lactate.

To test said inductive function, we are using a reporter-based assay system where psicA is used as a promoter to induce the transcription of a downstream red fluorescent protein gene (RFP). Characterization of pSicA involved harnessing TNFα as a signal to induce expression of the reporter and (Lou et al., 2019). The set-up involves fluorescence quantification of the TNFα inducible pSicA promoter system using a plate reader. We hypothesized that with increasing TNFα concentrations, we would expect to see a dose-dependent upregulation response of our engineered psicA-RFP construct. This result would corroborate results from Ma et al, 2010, which reported that TNFa upregulated sicA production, hence validating an inducible promoter in  S.enterica that would be indicative of TNFa-releasing immune cell presence.

## **Steps for Circuit Construction**

Our goal was to design a plasmid containing the psicA promoter with mRFP1 in a high copy number plasmid, pSB1C3, so that we can use it to transform it into Escherichia coli and S.enterica. To do this, we used the  Golden Gate Assembly (GGA) method to assemble our parts into a single plasmid by creating customized overhangs allowing assembly via their complementary overhang sequences (Engler et al., 2008).

**Repurposing pSBIC3 BBa\_J04450 to create a TNFa-inducible RFP plasmid**

We designed forward and reverse primers with overhang sequences to extract our desired part sequence of the pSBIC3 BBa\_J04450 plasmid. The pSBIC3 BBa\_J04450 plasmid construct contains the Lac operon which we cut out using PCR as described in the “Molecular cloning” section below. We analyzed our primer designs with IDT’s Oligo Analyzer. The calculated self-dimer values were below the recommended maximum delta F value of -9.0kcal/mole.

**Amplification of the psicA gblock fragment**

We ordered a Golden Gate compatible psicA gblock fragment which contained psicA with overlapping overhang sequences that allowed it to anneal with our repurposed “pSBIC3 BBa\_J04450 TNFα inducible RFP plasmid” that contain overhang sequences necessary for proper Golden Gate Assembly.

*Circuit Parts for psicA-RFP1 Construct Assembly*

| Final Golden Gate Assembled psicA-RFP Construct | ![](https://static.igem.org/mediawiki/2021/a/a0/T--British_Columbia--ExpV4.png) |

### **Construction of pLactate Characterization Circuit**

*Salmonella enterica* serovar Typhimurium has been shown to have  endogenous lactate utilization operons, orthologous to the lldPRD operon in *E. coli*, that are induced by the presence of L-lactate. Through this operon, *S. enterica* is able to use L-lactate generated by inflammation as an electron donor during gut colonization (Gillis et. al, 2019). Given *S. enterica*’s ability to self-regulate with L-lactate, we sought to build a bacterial reporter system from parts of the lactate-regulated operon to target the propensity for  lactate enrichment seen in many tumour microenvironments.

While the *E. coli lldPRD* operon has been well characterized, its homologous operon in *S. enterica* is poorly understood. Therefore, we needed to be able to characterize our construct containing the lactate regulatory units as a proof of concept before we could integrate it into the main split operon circuit. Similar to our psicA-TNFa system, we would activate the inducible promoter with varying concentrations of the induction molecule - in this case lactate - and  measure fluorescence of a downstream RFP.

## **How does *llPRD* operon work?**

This system behaves according to the negative regulation of the lactate promoter (pLactate). Since pLactate is a constitutively active promoter, the *lldR* genes are constantly being expressed. However, the regulatory LldR protein is also expressed in this system, which represses the activity of pLactate. In the presence of lactate, LldR is released from the pLactate promoter, allowing for activation of the promoter. Applied to our promoter proof-of-concept system, pLactate and LldR are expressed upstream of an RFP gene to be able to  correlate fluorescence to lactate-dependent promoter activity, hence controlling its expression in the presence of lactate. This construct allows us to perform lactate induction experiments to determine lactate dose-response curves at different lactate concentrations. Since lactate was chosen as our second biomarker for immune activity in the tumour microenvironment as described in on our [Project Design](https://2021.igem.org/Team%3ABritish_Columbia/Design) page, these assays allow us to validate the part’s efficacy at distinguishing healthy and tumour environments. With the information from the dose-response curves and literature on the different lactate concentrations in healthy tissue vs tumours, these assays allow us to validate the part’s efficacy at distinguishing healthy and tumor environments. With the information from the dose-response curves and literature on the different lactate concentrations in healthy tissue vs tumours,  we can establish if our biosensor can detect the range of lactate concentrations present in the TME and output an adequately strong response to be sensed in urine.

### **Circuit Construction of pLactate Reporter Construct**

The construct we wanted to build was:

![](https://static.igem.org/mediawiki/2021/e/e9/T--British_Columbia--LIP.png)

The pLactate and *lldR* sequences were obtained from the NCBI Genbank *Salmonella enterica* full genome sequence (Kroger, et. al, 2020). The *lldR* sequence was found by searching for the gene name in the database (between 3908879 - 3909655 bp). Because the pLactate sequence was not annotated, we needed to narrow down the sequence to be located between *lldP* and *sadA* (the regulatory region between the two genes). By cross referencing with the sequence of the *E. coli* lactate promoter, we were able to utilize the concept of gene conservation to pinpoint where the *S. enterica* lactate promoter was in the genome (3906960 - 3907177 bp). Because the pLactate sequence was not annotated, we needed to narrow down the sequence to be located between lldP and sadA (the regulatory region between the two genes). By cross referencing with the sequence of the E. coli lactate promoter, we were able to utilize the concept of gene conservation to pinpoint where the S. enterica lactate promoter was in the genome (3906960 - 3907177 bp).

The rest of the sequences were taken from biobricks. They are as follows: pSB1C3 vector, RFP (BBa\_E1010), Terminator (first; BBa\_J61102), Terminator (second, BBa\_B0015), (synthetic) Constitutive Promoter (BBa\_J23101), and RBS (BBa\_J61102).

## **Steps for Circuit Construction**

**1)** Using Benchling, IDT’s Oligochecker, and NCBI’s primer blast, we designed Golden Gate primers to make the parts compatible for assembly. Similarly, pLactate and lldR sequences were modified for Golden Gate Assembly by adding Golden Gate cut sites at the flanks of the sequences.

**2)** We ordered two gblocks, one with the lactate inducible promoter and RBS, and the other was a construct with the constitutive (synthetic) promoter (BBa\_J23101), RBS, *lldR*, and terminator. Parts were ordered with Golden Gate cut sites already incorporated. Primers were ordered for the PCR modification of RFP and the pSB1C3 backbone.

**3)** We ordered two gBlocks, one with the lactate inducible promoter and RBS, and the other was a construct with the constitutive (synthetic) promoter (BBa\_J23101), RBS, lldR, and terminator. Parts were ordered with Golden Gate cut sites already incorporated. Primers were ordered for the PCR modification of RFP and the pSB1C3 backbone.

### **Split-lux Operon Circuit Construction**

A split operon is a dual-plasmid system that serves as a genetic AND-gate. It is created by “splitting” an operon into different parts–two in our project–with each part encoded on a separate plasmid, typically under the control of a unique promoter. Independently, each part of the split operon is  nonfunctional. It is only when both parts of the operon are co-expressed, that the aggregate gene products from the different parts can carry out some function within the biological system.

![](https://static.igem.org/mediawiki/2021/a/a9/T--British_Columbia--UBC_lux_split_operon.png)

Our project involved splitting the lux operon, which is responsible for bioluminescence in many bacterial families, including Vibrionaceae, Shewanellaceae and Enterobacteriaceae (Brodl et al., 2018).
The genes expressed under the lux operon–and responsible for light production–are typically organized in the following order; LuxCDABEG.

## **Steps for Circuit Construction**

For our circuit construction, we designed and ordered gBlocks for each of our insert components, each containing pre-specified overhangs to allow for subsequent Golden Gate reactions. Using these gBlocks, two sets of Golden Gate reactions were carried out, similarly to the workflow described in the section about (psicA circuit construction), which was in accordance with the protocols in our Protocols page.

**Circuit 1** was assembled on the pSB1C3 backbone (found in the iGEM registry, and made Golden Gate compatible for BsaI digestion using PCR) — which includes the Chloramphenicol resistance gene.

**Circuit 2** was assembled on the pSB1K3 backbone (found in the iGEM registry, and made Golden Gate compatible for BsaI digestion using PCR) — which includes the Kanamycin resistance gene.

![](https://static.igem.org/mediawiki/2021/4/48/T--British_Columbia--UBC_circuit_plasmids.png)

### **Inducible Promoter Screening Vector Construction**

Circuit construction for the promoter discovery portion of the wet lab consists of a forward GFP and terminator as well as a reverse RFP and a reverse terminator assembled as shown below. The blunt end BamHI cut site in the middle will serves as the location of insertion of sheared *Salmonella* genomic DNA fragments.

## **Steps for Circuit Construction**

**1)** The functional DNA parts required to construct the screening vector could all be found in the iGEM distribution kit, hence we designed PCR primers to amplify the necessary parts to later be assembled via Golden Gate.

![](https://static.igem.org/mediawiki/2021/7/77/T--British_Columbia--UBC_promoter_trap_v3.png)

**2)** Molecular cloning would be carried out as described above in the psicA circuit construction section above.

**3)** Preparation of the screening construct for insertion of sheared *Salmonella* genome fragments involves production of blunt end fragments following a 2 hour 37 C digestion with BamHI. To blunt sticky ends after BamHI digestion, we would incubate the construct with T4 DNA polymerase and dNTPs for 30 minutes at 11 C as described in Bumann & Balvidia 2007. CIAP treatment after blunting would be used to reduce self-ligation. Resulting DNA fragments would then be used as described in our circuit characterization section for screening for other inducible promoters.

## **CIRCUIT VALIDATION**

---

### **psicA characterization**

A construct was set-up to characterize the psicA bioinformatically to elaborate  induction of the psicA promoter with TNFa, the protocol of the experimental set-up was adapted from Ma et al. 2010: shown below. Due to time constraints, a proof-of-concept experimental procedure could not be carried out, but the protocol is described:

## **A Proof-of-Concept Experimental Procedure**

Day before the experiment was run:

**1)** *Salmonella* cells with constitutively expressed RFP cells (positive control), cells with an empty plasmid (negative control), and cells with our psicA-RFP construct were inoculated at 37C overnight.

**2)** Non-agitated microaerophilic bacterial cultures will be prepared by inoculation of 5 ml of broth with 0.01ml of a stationary phase culture with varying concentrations of TNFa to form a dose response curve (i.e. Diluting TNFa solutions to 0ng/mL, 2ng/mL, 4ng/mL, 6ng/mL 8ng/mL, and 10ng/mL), followed by an overnight incubation (~18 h) at 37°C.

Day of the experiment:

**1)** Overnight cultures of bacteria will be concentrated 33-fold in Hank’s balanced salt solution (HBSS) supplemented with 10 mM HEPES, pH 7.4. The overnight cultures from the TNFa pretreated *Salmonella* strains will be washed thoroughly with HBSS three times to get rid of potential TNFa residue in the media. The bacteria will then be resuspended in fresh HBSS for cell lysis.

**2)** Next, cell-lysed bacteria will be loaded onto a 96 well plate using a multichannel pipettor, and fluorescence readout will be measured using plate reader.

Data would then be analyzed and graphed as was done for lactate induction experiments (see below and in our [Results](https://2021.igem.org/Team%3ABritish_Columbia/Results) page). If the promoter would work as expected, we would observe increasing levels of RFP expression as TNFa concentration increases.

### **pLactate characterization**

As the lactate inducible promoter gets induced once lactate binds to its substrate, we measured the promoter's activity by adding different concentrations of lactate in a plate reader (Goers et al, 2017)

## **Experimental Procedure**

Day before the experiment was run:

**1)** *Salmonella* cells with constitutively expressed RFP cells (positive control), cells with an empty plasmid (negative control), and cells with our pLactate-LldR-RFP construct were inoculated at 37C overnight.

**2)** We prepared the lactate media the day prior. Lactate solutions were diluted to the appropriate concentrations to be put in the reader (0mM, 2mM, 5mM, 15mM, 40mM and 100mM lactate). These concentrations were determined through literature searches of lactate presence in healthy and tumourous human tissue.

Day of the experiment

**1)** The OD600 of the cells was diluted to 0.5 OD600 (and checked using the spectrometer) so that the concentration of the cells is correct after being added into the media by the liquid dispenser.

**2)** The lactate media with cells was loaded into a 96 plate using a multichannel pipettor, and fluorescence readout measured using plate reader.

From this assay we hoped to validate the promoter’s nature, as it is induced by different lactate concentrations. If the promoter would be working as expected, we would be able to see a greater RFP expression as lactate concentration increases, and consequently prove that the promoter worked. After running the experiment and collecting the data, we were able to see the promoter getting induced by seeing increasing levels of RFP being expressed as concentration of lactate increased, successfully achieving a proof-of-concept experimental results. To see the figures and explanation of results from this assay, please head over to our [Results](https://2021.igem.org/Team%3ABritish_Columbia/Results) page or [Engineering Success](https://2021.igem.org/Team%3ABritish_Columbia/Engineering) page.

### **Functional Validation of the Split Operon**

In our future experiments, we are planning to validate the functionality of our split operon. After the simultaneous electroporation of the lactate and TNFa inducible plasmids with the split operon to *Salmonella*, the constructs would be plated on a LB medium plate with chloramphenicol and kanamycin antibiotics. This will ensure that only the bacteria which took up both of the plasmids are growing on the plate. The colonies would be picked the next day and genotyping PCR would be performed to make sure the construct is present. Successful colonies would be inoculated and confirmed once again for successful transfection with restriction digest check with EcoRI and PstI. Upon confirmation of the successful transfection, glycerol stock would be prepared for future use of the constructs.

Next, the bacterial cells will be subjected to various treatments and luciferase light output will be measured to confirm the functionality of the both circuits. After the pSicA-RFP and pLactate-RFP characterization experiments, the optimal concentration for promoter induction will be elucidated. This optimal concentration will be used to induce simultaneous promoter induction in *Salmonella* with both plasmids present. The following conditions will be tested:

* *Salmonella* transfected with pSicA-LuxABG and pLactate-LuxCDE with **optimal concentrations of lactate and TNFa**.
* *Salmonella* transfected with pSicA-LuxABG and pLactate-LuxCDE with optimal concentrations of lactate and **no TNFa.**
* *Salmonella* transfected with pSicA-LuxABG and pLactate-with **no lactate** and optimal concentrations of TNFa.
* *Salmonella* transfected with split LuxABG and LuxABG plasmids under the control of  **the constitutive promoters** (Positive Control).
* *Salmonella* transfected with **empty** pSB1C3 and SB1K3 backbones (Negative Control).

Concentrations will be changed to lower or higher than optimal concentrations as needed, if no or little luciferase output is detected in the plate reader. Experiments will be performed in triplicates to ensure statistical significance.

Given that the system that is relevant for implementation of this model is human, we would like to explore validation in hydrogel tumor microenvironment (TME) reconstructions. However, beyond this, another important avenue we would like to explore is hydrogel tumour microenvironment (TME) reconstructions. 2D tumour models, like cell lines, poorly mimic the extracellular matrix (ECM) and spatial distribution around the TME, so 3D models are preferred when exploring the intricate crosstalk of cells around the tumour (Fernando et al., 2021). Recently, hydrogel-based 3D TME recapitatilation has proved itself as an excellent resource for in-vitro tumour modelling (Rodrigues et al., 2021). Hydrogels are networks of cross-linked polymers that are “swollen” by water; they are a great in vitro model, as they have the capacity to recapitulate ECM, model protein adhesion and sequesterilization (Caliari et Burdick, 2016). As a part of our Integrated Human Practices efforts, we have interviewed [Dr. Anne-Marie Fortier from McGill](https://2021.igem.org/Team%3ABritish_Columbia/Human_Practices), who works with hydrogel for precision oncology and kindly consulted us on hydrogel experimental protocol optimization. We have concluded that for our purposes, Polyethylene Glycol (PEG) hydrogel will be optimal. We will use the hydrogel as a biological scaffold to seed with murine TME cells. The cells will be requested from UBC labs that are investigating tumourigenesis in a murine model and are willing to share the TME cell suspensions from hot and cold tumours. The following protocol will be implemented:

## **Experimental Procedure**

**1)** To add peptide sequences to polymer, react peptide of interest with DA-PEG-NHS in 50mM Sodium Bicarbonate (pH 8.5) for 2hrs. Peptide:PEG = 1:1 molar ratio.

*Final concentration of peptide on PEG hydrogel should be between 1.4-5 mM*

For 100 uL PEG hydrogel, 3 mM RGDS, 0.13 mg RGDS (65 uL of 2 mg/mL stock), 1.02 mg PEG-NHS

**3)** Lyophilize mixture (O/N) and store frozen, protected from light.

**4)** Combine 0.2g/ml PEGDA+PEG (in desired ratio and total weight percent for mechanical compliance) and 0.3mmol/ml triethanolamine in 50mM Tris (pH 8.5).

For 100 uL (final) of PEGDA solution: 20 mg PEGDA+PEG, 2.8 mg triethanolamine-HCl (F.W. 185.65), 100 uL 50mM Tris

**5)** Add acryloyl-PEG-RGDS and photoinitiator (4 uL into 100 uL polymer of 2,2-dimethyl-2-phenyl-acetophenone (DPA) in n-vinylpyrrolidone (VP) at 600 mg/mL).

**6)** Sterilize entire polymer solution through 0.2um filter into appropriate tissue culture dish.

**7)** Add cell suspension to this polymer solution to create cell-polymer solution with a cell density of 3-5 x 106 cells/ml.

**8)** Photopolymerize with UV light (1-10 min).

**9)** At this point, 3-D constructs can be transferred to larger well-dish and surrounded with growth media to incubate.

Upon the seeding of the hydrogel, we plan to co-culture it with the *Salmonella* transfected with pSicA-LuxABG and pLactate-LuxCDE as per the Johnston et al. protocol. *Salmonella* will then be isolated and inspected under the plate reader for luciferase activity.

## **INDUCIBLE PROMOTER DISCOVERY**

---

To hunt for inducible promoters in the *Salmonella* genome, we implemented a promoter trap adapted from the design from [UBC iGEM 2019](https://2019.igem.org/Team%3ABritish_Columbia) and Bumann and Valdivia (2007). In a promoterless vector, we inserted a restriction site (BamHI) upstream of a forward reporter (GFP) and a reverse reporter (RFP). The restriction is where fragments from the sheared *Salmonella* genome will be inserted. If this inducible promoter happens to have directionality and regulates in the forward direction, it will drive the expression of GFP, and if it regulates the reverse direction, it will drive the expression of the reverse RFP. These results can be sorted by colour, resulting in candidate inducible promoters that can be further validated through sequencing.

Even though we did not reach the assay development stage of this section of our project, we have outlined how we would carry these assays out. To produce the sheared *Salmonella* genome, we will follow the protocol outlined in Bumann and Valdivia (2007), involving the following procedures:

## **Experiment design of shearing Salmonella genome**

**1)** Genomic isolation from untransformed *Salmonella* SL1344 ∆invA∆ssaR.

**2)** Gel electrophoresis of genomic isolation products to confirm that genomic DNA is not smeared or cleaved.

**3)** Sonication of bacteria in 10 second intervals for 30 seconds, then transferring 4ul aliquots of DNA from the sonication to a 1.5% gel to observe size of fragments. Sonication will be repeated until gel shows fragments of 500-700bp. This length is large enough to generously capture promoter and regulatory elements of the genome.

**4)** Gel purification of gel segments from the sonication that resulted in 500-700bp fragment.

![](https://static.igem.org/mediawiki/2021/2/2f/T--British_Columbia--UBC_iGEM_sheared_genome_expected.png)
Expected results of sheared genome

Once our genomic fragments have been produced, 20ul ligations to our previously BamHI-digested construct will ensue, leaving at 4C overnight. To screen for a regulatory element, we will produce a stable line of *Salmonella* strain SL1344 ∆invA∆ssaR transformed with the screening construct, whose colonies will be kept as glycerol stocks in -80C aliquots for future use. From a streaked iteration of this line, we will discard any fluorescent colonies through cell sorting since they would be demonstrating non-specific fluorescence that fails to be regulated by our screening construct.

Since induction of psicA by TNFa has been reported (Ma et al, 2010) but occurs through an unknown mechanism, hence decreasing the reliability of this correlation to be used by other researchers, we decided to screen for other TNFa-inducible promoters in *Salmonella* genome. Additionally, obtaining psicA in our screening results from this assay would serve as a proof-of-concept that our assumption about the relationship between TNFa and psicA is correct. For this same reason, we chose to test for induction with lactate as a proof-of-concept as well. Since hypoxia is a key characteristic of the tumour microenvironment and a feature that naturally localizes *Salmonella* to tumours in the first place (Chien et al, 2017), we decided to also screen for promoters that are inducible by hypoxia through incubating *Salmonella* with our screening vector in an anaerobic chamber previous to cell sorting. Even though we would initially screen for inducible promoters using these two conditions, we would expand to other conditions once these were completed as proof-of-concept.

Cell sorting would be carried out using a *Salmonella* strain SL1344 ∆invA∆ssaR transformed with an empty vector as a negative control, and transformed with a constitutively expressed RFP as a positive control. Sorting would be based on fluorescence levels of both green and red from reverse RFP and forward GFP expression, respectively. Once sorted, cells with highest fluorescence would be grown overnight with the same volume of its induction molecule (or in the hypoxic chamber), and sorted in the same manner the next day to ensure induction was not a false positive. Cells that have been selected on both rounds of sorting will be plated, and plasmid DNA purified to further characterize candidate gene fragments that induced fluorescence.

## **References**

**[1]** Fernando, K., Kwang, L. G., Lim, J. T. C., & Fong, E. L. S. (2021, April 7). Hydrogels to engineer tumor microenvironments: In vitro. Biomaterials Science. Royal Society of Chemistry. https://doi.org/10.1039/d0bm01943g

**[2]** Rodrigues, J., Heinrich, M. A., Teixeira, L. M., & Prakash, J. (2021, March 1). 3D In Vitro Model (R)evolution: Unveiling Tumor–Stroma Interactions. Trends in Cancer. Cell Press. https://doi.org/10.1016/j.trecan.2020.10.009

**[3]** Caliari, S. R., & Burdick, J. A. (2016, May 1). A practical guide to hydrogels for cell culture. Nature Methods. Nature Publishing Group. https://doi.org/10.1038/nmeth.3839

**[4]** Johnston, T. G., Yuan, S. F., Wagner, J. M., Yi, X., Saha, A., Smith, P., … Alper, H. S. (2020). Compartmentalized microbes and co-cultures in hydrogels for on-demand bioproduction and preservation. Nature Communications, 11(1). https://doi.org/10.1038/s41467-020-14371-4

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# Team:British Columbia/Results

UBC iGEM 2021

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EXPERIMENTS

# Results

[Construction of psicA](#psicA)

---

[Construction of pLactate](#pLactate)

---

[Characterization of pLactate](#characterization)

## **CIRCUIT CONSTRUCTION OF psicA**

---

Circuit construction of psicA-RFP was carried out as described on our [Experimental Design](https://2021.igem.org/Team%3ABritish_Columbia/Experiments) page. Below are results of diagnostic experiments (agarose gels) to confirm the size of our inserts and plasmid.

**Repurposing BBa\_J04450 plasmid to create a Golden Gate compatible psicA-RFP construct:**

![PCR amplification of psicA gblock fragment (143bp)](https://static.igem.org/mediawiki/2021/6/6f/T--British_Columbia--psicA_PCR_amp.png)

PCR amplification of psicA gblock fragment (143bp)

**Figure 1.** PCR amplification of the psicA gblock was successful. In lane 1, the NEB 1 kb plus DNA ladder is observed. Lanes 2 and 3 contain the amplified psicA gblock. Lane 3 is a replicate amplification. As labelled in the figure, the DNA ladder confirms the 143bp size of the psicA gblock. This confirms the presence of the psicA gblock part in preparation for Golden Gate assembly of the construct.

![PCR amplification of mRFP1 reporter gene from BBa_J04450 plasmid  (2969bp)](https://static.igem.org/mediawiki/2021/4/40/T--British_Columbia--UBC_RFP_PCR.png)

PCR amplification of mRFP1 reporter gene from BBa\_J04450 plasmid (2969bp)

**Figure 2.** PCR amplification of the mRFP1 reporter gene containing BBa\_J04450 plasmid was successful. In lane 1, the NEB 1 kb plus DNA ladder is observed. Lanes 2 and 3 contain the amplified BBa\_J04450 plasmid backbone. Lane 3 is a replicate amplification. As labelled in the figure, the DNA ladder confirms the 2969bp size of the plasmid backbone. This confirms the presence of the backbone in preparation for the Golden Gate assembly reaction with the amplified psicA gblock.

![Expected PCR amplification results using Benchling DNA Digest Tool](https://static.igem.org/mediawiki/2021/5/5b/T--British_Columbia--BenchlingDigest.png)

Expected PCR amplification results using Benchling DNA Digest Tool

**Figure 3.** The mock restriction digest feature in the virtual Benchling tool for visualization of DNA plasmids was utilized to provide expected results for a real restriction digest of our Golden Gate assembled plasmid. It demonstrates that in a correctly assembled plasmid, a restriction digest using the EcoR1 restriction enzyme at the EcoR1 recognition sites along the BBa\_J04450 plasmid produces a 3082bp long fragment as seen in lane 3 (visualized using Life 1kb Plus DNA ladder). Taken together with Figures 1 and 2, it provides expected agarose gel results for a restriction digest performed on the Golden Gate assembled plasmid containing the psicA gblock and mRFP1 reporter gene.

## **CIRCUIT CONSTRUCTION OF pLactate**

---

Circuit construction of pLactate-RFP was carried out as described on our [Experimental Design](https://static.igem.org/mediawiki/2021/3/33/T--British_Columbia--pLactate_gel.jpg) page. Below are results of diagnostic experiments (agarose gels) to confirm the size of our inserts and plasmid.

![gel to validate pLactate circuit construct](https://static.igem.org/mediawiki/2021/3/33/T--British_Columbia--pLactate_gel.jpg)

Gel to validate pLactate circuit construct

**Figure 4.** The uncut lane was an uncut control (uncut plasmid), displaying what the samples would look like if the digestion failed. Lane 3(1) was our construct plasmid cut with 1 enzyme. It hovers around the 4.5 kb mark as expected. Lane 3(2) was our circuit construct cut with 2 predetermined enzymes. As you can see, even though one of the plasmids didn’t completely cut the plasmid, the second band hovering above the 2kb mark indicates that when both enzymes are actively cutting the plasmid, it gives us DNA fragments of around ½ the size of the original construct. This also helps validate that the size of the construct is correct.

![Sanger sequencing results](https://static.igem.org/mediawiki/2021/2/25/T--British_Columbia--SangerSeq.png)

**Figure 5.** Above are the results of our Sanger sequencing. All of our primers worked, and the sequences aligned to our designed construct, fully confirming that we have what we set out to assemble.

## **LACTATE-INDUCIBLE PROMOTER CHARACTERIZATION**

---

### **Iteration1: Characterizing in *E. coli***

Induction experiments with differing concentrations of lactate were carried out on our constructed pLactate following circuit construction as described in [Experimental Design](https://2021.igem.org/Team%3ABritish_Columbia/Experiments). The results of this characterization experiment are described below.

![LIP Experiment Plots](https://static.igem.org/mediawiki/2021/2/22/T--British_Columbia--LIP_plots.jpg)

**Figure 6A. Data from our lactate induction experiment with our lactate-inducible reporter construct, with the OD600 (cell density) plotted over time.** We discarded the 0h timepoint data due to technical artifacts caused by bubbles in the plate reader wells leading to a spike in the OD600 measurements. Looking at the 2h time point (second read), the bubbles have disappeared, removing the obstruction towards measuring the OD600 of the cells. Cell growth in media with a lactate concentration of 100 mM is noticeably lower than the rest of the concentrations. This matches the literatures’ reports of lactate poisoning at higher concentrations in *E. coli*.

**Figure 6B and 6C. Data from our lactate induction experiment with our lactate-inducible reporter construct, with the lactate concentrations plotted against normalized fluorescence.** As expected, 0h has the lowest fluorescence, especially since the cells haven’t had time to acclimate to the presence of lactate in the media. Overall, there is a pattern of increased fluorescence with an increase of lactate concentration, with a particularly steep increase in the concentrations between 0 and 25 mM seen in plot C. Unsurprisingly, the steep increase at the lower concentrations match the kinetics of a similar lactate operon found in *E. coli* which mirrors a Hill function. The fluctuations between hours may be due to technical artifacts and the large 2 hour time span between each read, especially at hour 2. However, as seen in plot B, there is still an overarching increase of fluorescence over time with most of the times later in the experiments expressing higher amounts of fluorescence.

### **Iteration2: Characterizing in *Salmonella***

![LIP Experiment Plots](https://static.igem.org/mediawiki/2021/b/b2/T--British_Columbia--LIP_salm.png)

**Figure 7. Relative fluorescence and predicted luminescence from the *Lux* operon produced under different concentrations of lactate over time.** (A) Relative fluorescence overtime. (B) Predicted luminescence overtime.

Measurement was taken every ten minutes over a course of 13 hours. 11 different lactate concentrations were tested, as indicated by the colour coding. Relative fluorescence was calculated by normalizing against OD600 and the highest fluorescence detected in the entire experiment. At the initial six hours, fluorescence level was relatively stable, indicating a lag phase. Afterwards, in media with 40mM and 100mM lactate, fluorescence increased as time went on, whereas it stabilized or decreased in other lactate concentrations.

As for light output, it was predicted under 40mM and 100mM, there would be different lactate concentrations. It is suited for our need as tumour lactate concentration is typically higher. It is also predicted that after six hours, the light production would be stabilized.

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###### Project

[Description](https://2021.igem.org/Team%3ABritish_Columbia/Description)

[Design](https://2021.igem.org/Team%3ABritish_Columbia/Design)

[Implementation](https://2021.igem.org/Team%3ABritish_Columbia/Implementation)

[Partnerships](https://2021.igem.org/Team%3ABritish_Columbia/Partnership)

###### Experiments

[Experiments](https://2021.igem.org/Team%3ABritish_Columbia/Experiments)

[Engineering](https://2021.igem.org/Team%3ABritish_Columbia/Engineering)

[Results](https://2021.igem.org/Team%3ABritish_Columbia/Results)

[Parts](https://2021.igem.org/Team%3ABritish_Columbia/Parts)

[Contributions](https://2021.igem.org/Team%3ABritish_Columbia/Contribution)

[Protocols](https://2021.igem.org/Team%3ABritish_Columbia/Protocols)

[Lab Notebook](https://2021.igem.org/Team%3ABritish_Columbia/Notebook)

[Safety](https://2021.igem.org/Team%3ABritish_Columbia/Safety)

###### Models

[Mathematical Modelling](https://2021.igem.org/Team%3ABritish_Columbia/Model)

[Software Simulations](https://2021.igem.org/Team%3ABritish_Columbia/Model)

###### Human Practices

[Integrated HP](https://2021.igem.org/Team%3ABritish_Columbia/Human_Practices)

[Outreach](https://2021.igem.org/Team%3ABritish_Columbia/Communication)

[Education](https://2021.igem.org/Team%3ABritish_Columbia/Education)

[Collaborations](https://2021.igem.org/Team%3ABritish_Columbia/Collaborations)

[Book Chapter](https://2021.igem.org/Team%3ABritish_Columbia/Book_Chapter)

###### Team

[Members](https://2021.igem.org/Team%3ABritish_Columbia/Team)

[Attributions](https://2021.igem.org/Team%3ABritish_Columbia/Attributions)

[Sponsors](https://2021.igem.org/Team%3ABritish_Columbia/Sponsors)