iGEM 2019 — Team Nantes

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# Team:Nantes/Notebook

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[Judging form](https://igem.org/2019_Judging_Form?team=Nantes)

Awards

[Medals](https://2019.igem.org/Team%3ANantes/Medals)

[Awards](https://2019.igem.org/Team%3ANantes/Awards)

Achievements

[Results](https://2019.igem.org/Team%3ANantes/Results)

[Model](https://2019.igem.org/Team%3ANantes/Model)

[Collaborations](https://2019.igem.org/Team%3ANantes/Collaborations)

[Parts](https://2019.igem.org/Team%3ANantes/Parts)

[Demonstrate](https://2019.igem.org/Team%3ANantes/Demonstrate)

[Improve](https://2019.igem.org/Team%3ANantes/Improve)

Human Pratices

[Integrated](https://2019.igem.org/Team%3ANantes/Human_Practices)

[Education-Engagement](https://2019.igem.org/Team%3ANantes/Public_Engagement)

Project

[Description](https://2019.igem.org/Team%3ANantes/Description)

[Design](https://2019.igem.org/Team%3ANantes/Design)

[Experiments](https://2019.igem.org/Team%3ANantes/Experiments)

[Notebook](https://2019.igem.org/Team%3ANantes/Notebook)

[Safety](https://2019.igem.org/Team%3ANantes/Safety)

[Attributions](https://2019.igem.org/Team%3ANantes/Attributions)

Team

[Team](https://2019.igem.org/Team%3ANantes/Team)

[Sponsors](https://2019.igem.org/Team%3ANantes/Sponsors)

Notebook
Notebook

## Notebook PDF


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# Team:Nantes/Experiments

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[Judging form](https://igem.org/2019_Judging_Form?team=Nantes)

Awards

[Medals](https://2019.igem.org/Team%3ANantes/Medals)

[Awards](https://2019.igem.org/Team%3ANantes/Awards)

Achievements

[Results](https://2019.igem.org/Team%3ANantes/Results)

[Model](https://2019.igem.org/Team%3ANantes/Model)

[Collaborations](https://2019.igem.org/Team%3ANantes/Collaborations)

[Parts](https://2019.igem.org/Team%3ANantes/Parts)

[Demonstrate](https://2019.igem.org/Team%3ANantes/Demonstrate)

[Improve](https://2019.igem.org/Team%3ANantes/Improve)

Human Pratices

[Integrated](https://2019.igem.org/Team%3ANantes/Human_Practices)

[Education-Engagement](https://2019.igem.org/Team%3ANantes/Public_Engagement)

Project

[Description](https://2019.igem.org/Team%3ANantes/Description)

[Design](https://2019.igem.org/Team%3ANantes/Design)

[Experiments](https://2019.igem.org/Team%3ANantes/Experiments)

[Notebook](https://2019.igem.org/Team%3ANantes/Notebook)

[Safety](https://2019.igem.org/Team%3ANantes/Safety)

[Attributions](https://2019.igem.org/Team%3ANantes/Attributions)

Team

[Team](https://2019.igem.org/Team%3ANantes/Team)

[Sponsors](https://2019.igem.org/Team%3ANantes/Sponsors)

Protocols & Experiments
Protocols & Experiments

![](https://static.igem.org/mediawiki/2019/0/09/T--Nantes--protocolsandexperimentsimg.jpg)

[![](https://static.igem.org/mediawiki/2019/2/2b/T--Nantes--pcr.jpg)](#pcr)

[![](https://static.igem.org/mediawiki/2019/1/1b/T--Nantes--gibson.jpg)](#gibson)

[![](https://static.igem.org/mediawiki/2019/2/2c/T--Nantes--quickchangepcr.jpg)](#quickchange)

[![](https://static.igem.org/mediawiki/2019/8/87/T--Nantes--electro.jpg)](#electro)

[![](https://static.igem.org/mediawiki/2019/3/35/T--Nantes--digestion.jpg)](#digestion)

[![](https://static.igem.org/mediawiki/2019/d/d0/T--Nantes--spectrofluo.jpg)](#spectrofluo)

[![](https://static.igem.org/mediawiki/2019/7/74/T--Nantes--miniprep.jpg)](#miniprep)

[![](https://static.igem.org/mediawiki/2019/f/f7/T--Nantes--competence.jpg)](#competence)

[![](https://static.igem.org/mediawiki/2019/0/0e/T--Nantes--fixation.jpg)](#fixation)

[![](https://static.igem.org/mediawiki/2019/4/49/T--Nantes--transformation.jpg)](#transformation)

## PCR

##### Material :

* P10, P100
* Tubes PCR (O.2 mL)
* Thermocycler
* Sterile water
* 5x PHUSION HF
* DNTPs (10uM)
* Forward Primer (10uM)
* Primer Reverse (10uM)
* Diluted DNA (50mg/uL)
* PHUSION DNA Pol (2U/uL)

##### Protocol :

* Add in a PCR tube (total volume = 50uL) :
  + 32.5 uL of dilluted water
  + 10 uL 5x PHUSION HF
  + 1 uL of dNTP
  + 2.5 uL of Forward Primer
  + 2.5 uL of Reverse Primer
  + 1 uL diluted DNA
  + 0.5 uL PHUSION DNA Pol
* Place the tube in the thermocycler and start the programm
  associated with the DNA used as a matrix
* Store the result at -20°C (-4°F)

## Gibson

Goal: Assemble the different DNA parts recuperated by PCR

##### Material :

* PCR tube
* Purified DNA
* Pipette p10
* Hot plate (50°C/122°F)
* Gibson reactant
* Vortex
* RNase-free water

##### Protocol :

* Deposit the adapted volumes of purified DNA in a PCR tube
* Vortex quickly
* Add 5uL of the Gibson reactant
* Mix the solution by pipetting back and forth
* Incubate at 50°C for 1h
* Store the tube at -20°C (-4°F)

## PCR Quick Change

##### Protocol :

* Add into a 50uL PCR tube
* 36uL of distilled water
* 10uL of 5x reaction buffer
* 1uL (we want to have 100 ng of DNA in a total volume of 50uL) of plasmid pSRL from Guy
* 1uL (125 ng) of primer #1 (forward Quick Change)
* 1uL (125ng) of primer #2 (reverse Quick Change)
* 1 uL of dNTP
* Just before the PCR add 1uL of pHusion
* Program x20

|  |  |
| --- | --- |
| Temparature | Times |
| 95°C | 30 sec |
| 95°C | 30sec |
| 55°C | 1min |
| 72°C | 6min |
| 72°C | 10min |
| 14°C | infinity |

* Add 1uL of DPN1 incubate 1h at 37°C

## Incubate of the PCR Quick Change with DPN1

Goal : This enzyme degrades the DNA matrix

##### Protocol :

* Add 1 uL of DPN1 (in cold conditions!! Because of the presence of enzyme)
* Incubate at 37°C for 1h in a PCR machine
* Inactivate the enzyme at 80°C for a few minutes

## Electrophoresis

##### Material :

* pipettes p100, p20, p10
* Load buffer
* Size marker
* Electrophoresis machine
* PCR results
* Centrifugation tubes
* Electrophoresis gel

##### Protocol :

* The loading buffer needs to be at a concentration of 1x in samples of 50uL :
  add 10uL to 50uL of PCR result
* Preparation of 20 uL of size markers with loading buffer :
  + 1 uL of size markers x 5
  + 1 uL of loading buffer x 5
  + 4 uL of water x 5
* Deposit 6 uL of the marker+loading buffer solution into 1 gel pit
* Deposit 60 uL of each sample into 2 pits (30 uL in each)
* Once the migration is finished, observe the results with UV rays. Be very cautious to respect all security guidelines. Minimise the DNA’s exposition to UV rays because the longer the DNA is exposed the higher the risk that it will be distorted
* Cut out the pieces of gel that contain the DNA that migrated as wanted and store each piece in a centrifugation tube at -20°C (-4°F)

## Digestion

Goal : Recuperate pieces of certain plasmids

##### Material :

* H2O
* NE Buffer
* EcoR1 HF
* Pst1
* Spe1
* Xba1

##### Protocol:

* **Vector** : Add in the same tube (total volume = 50uL) :
  + 5 uL NE Buffer 2.1
  + X uL plasmid (1ug) (1000ng/50uL = 20ng/uL so : (20 x 50)/“concentration Nanodrop obtained from the mini prep” = XuL)
  + 1 uL of Eco RI HF
  + 1 uL of Pst1
  + Complete to 50uL with distilled water
* **Insert :**
  + 1) Digestion EcoR1 HF / Spe 1
    - 5uL of NE buffer 2.1
    - X uL of Insert (500 mg) (500ng/50uL = 10ng/uL so 10x50/“Nanodrop concentration obtained Miniprep” = X uL)
    - 1 uL of Eco R1 HF
    - 1 uL Spe1
    - Complete to 50uL with distilled wate
  + Digestion Xba1/Pst1
    - 5uL of NE Buffer
    - (Same formula as before) XuL of insert (500mg)
    - 1 uL of Xba1
    - 1 uL of Pst1
    - Complete to 50 uL of H2O

    |  |  |  |  |  |
    | --- | --- | --- | --- | --- |
    | Enzyme/Insert | pLAC | pARA | pSRL | pRIB |
    | EoR1-Spe1 | x | x | x |  |
    | Xba1-Pst1 |  | x | x | x |

    - Incubate reaction at 37°C for 30 min
    - Inactivate 80°C for 20 minutes

## Spectrofluorometry

Goal : Measure the Optical Density and the fluorescence of transformed bacteria in different mediums

##### Material :

* Tecan
* Bacterial Pre-Culture in
* Pipette p100
* Black plates with 96 pits (to measure fluorescence)
* Transparent plates with 96 pits (to measure Optical Density)
* Bunsen Burner

##### Protocol:

* Deposit 143 uL of appropriate sugar medium, 7 uL of appropriate bacterial preculture and 50 uL of mineral oil into each plate
* Measure the OD and fluorescence with the TECAN

## Mini-preps (Kit EZNA -> Plasmid DNA mini kit I Quick Guide) provided by Omega

##### Material :

* Centrifuge
* Heating plate
* Steril water
* Reactants from the kit
* P100 + cones
* Nanodrop

##### Protocol :

* From a bacterial pre-culture in 5 mL LB
* Centrifugate at 7850 rpm for 2min30sec at room temperature 21°C
* Eliminate the supernatant by turning the tube upside down in a biological liquid trash
* Add 250 uL of the solution I and resuspend the pellet with the pipette
* Transfer into an eppendorf tube of 2 mL
* Lysis step : Add 250 uL of the solution II and mix cautiously by turning the tube upside down 1 or 2 times. Careful the bacteria are very fragile at this point
* Wait around 1 minute until the solution becomes slimy. Do not go over 2 minutes!
* Add 350 uL of the solution III and mix by turning the tube upside down and back until « white flakes » appear. Centrifugate at 11180 rpm during 15 minutes
* Insert the column in the collector tube.\*
* Deposit the supernatant from the centrifugation in the column (Very sensitive step, be very careful)
* Centrifugate for 1 min at maximum intensity
* Add 500 uL of HBC buffer
* Centrifugate at 11 180 rpm for 1 minute then eliminate the supernatant
* Add 700 uL of DNA wash buffer
* Centrifugate at 11 180 rpm for 1 minute then eliminate the supernatant
* Add 700 uL of DNA was buffer
* Centrifugate at 11 180 rpm for 1 minute then eliminate the supernatant
* Centrifugate the open tubes at 11 180 rpm for 2 minutes (this allows the column to dry
* Deposit the column in an eppendorf tube of 1.5 mL and let dry for 10 min (open tube)
* Add 50 uL of hot sterile water and leave it to sit at room temperature for 1 minute
* Centrifugate at 11 180 rpm for 1 minute
* Measure the concentration and purity of the DNA with the Nanodrop
* Store DNA at -20°C

## Competence with TSS

Make bacteria competent, meaning to weaken their cell wall to facilitate the entry of plasmids inside the bacteria. This method is preferred on bacteria that were stocked beforehand

##### Material :

* Spectrophotometer

##### Protocol :

* Seed 25 mL of LB with 500 uL of pre-culture
* Incubate at 37°C in an agitator. Monitor the growth by measuring the Optical Density at 550 nm. Stop the culture when the OD is between 0.45 and 0.5
* Place the eppendorf tubes on ice for 10 minutes. Make aliquots of the culture of 1 mL in the tubes
* Incubate the DNA/competent bacteria mix on ice for 30 minute
* Centrifugate at **4000 G** for 4 to 5 minutes at 4°C
* Eliminate the supernatant in the biological liquid trash
* Delicately resuspend (without agitating or pipetting) the pellets in 100 uL do cold TSS at 4°C
* Store the tubes at -80°C

## Test competence with transformation

##### Material :

* Petri Dishes containing LB agar and antibiotic
* Plasmid used to transform (pET11A)
* Hot plate
* Crushed ice
* LB
* Centrifuge
* Agitator/Incubator

##### Protocol :

* Put 1 uL of plasmid « pET11A » (90 ng of DNA) in an eppendorf tube containing the « competent » bacteria to be tested
* Incubate for 30 minutes on ice
* Thermal shock : incubate 45 seconds at 42°C then on ice for 2 minutes
* Add 800 uL of LB
* Incubate for 1 hour under agitation at 100 rpm
* Centrifugate 5 minutes at **3000 G**
* Remove the supernatant and resuspend the pellet with 100 uL of this supernatant
* Spread 100 uL of this solution on a Petri dish containing LB agar and the associated antibiotic
* Incubate in oven at 37°C overnight
* See if their are any colonies the next day

## Competence with TB+

Make bacteria competent, meaning to weaken their cell wall to facilitate the entry of plasmids inside the bacteria. This method is preferred for bacteria that have been freshly grown

##### Material :

* In cold chamber
* Bacterial pre-culture
* LB
* Spectrophotometer
* Sterile eppendorf tube of 1.5 mL
* 2 falcon tubes of 50mL
* Crushed ice
* Pipette de 25mL and 50mL
* Pipette p1000

##### Protocol :

* From a bacterial preculture in 10mL of LB with antibiotics
* Dilute the preculture 1/100
* Incubate at 37°C with agitation
* Follow the Optical Density at 550nm (blank = 650 uL of LB with antibiotics)
  Careful! Stop the culture when the OD is between 0.45 and 0.5 to have the right amount of bacteria
* **In the cold chamber**
  + Separate the previous pre-culture in 2 eppendorf tubes of 50 mL (cold)
  + Incubate 10 minutes on ice
  + Centrifugation 10 to 15 minutes at 2500 rpm at 4°C
  + Eliminate the supernatant
  + Cautiously resuspend the pellet in 2mL (no vortex or aspiration/respiration with the pipette)
  + Mix carefully
  + Incubate 10 min on ice
  + Reassemble both tubes in 1 tube
  + Add DMSO at 7%
  + Mix carefully
  + Make aliquots of 100 uL of the competent bacteria (in eppendorf tubes of 1.5mL)
  + Store at -80°C

## Bacterial fixation with PFA

Goal : Immediate stop bacterial growth at a precise moment

##### Material :

* Incubator/Agitator
* Bacterial pre-culture
* Spectrophotometer
* Centrifuge
* Eppendorf tubes
* Pipettes P1000, P200, P10
* PFA at 4%
* PBS (1x)
* Crushed ice

##### Protocol :

* From the bacterial pre-culture take 1 mL and measure the Optical Density at 550nm
* From this same preculture take 1mL and place in an eppendorf tube (Triplicate samples) :
  + Centrifugate at **10000 G** for 5 minutes
  + Eliminate the supernatant
  + Add 100 uL of PFA at 4%
  + Incubate 10 to 15 minutes on ice
  + Centrifugate at **10000 G** for 5 minutes
  + Eliminate the supernatant (in a toxic PFA trash)
  + Add 100 uL of PBS
  + Centrifuge at 10000G for 5 minutes
  + Eliminate the supernatant
  + Add 100 uL of PBS
  + Centrifugate at 10000G for 5 minutes
  + Eliminate the supernatant
  + Add 400 uL of PBS
  + Store at 4°C

## Bacterial transformation

Goal: Genetically modify bacteria by adding one or many genes

##### Material :

* Agitator/Incubator at 37°C
* Water bath at 42°C
* Crushed Ice
* Centrifugation tubes or 1.5mL tubes
* Autoclave
* LB medium
* Petri dish containing LB agar and antibiotic (Ampicillin or Kanamycin depending
  on the plasmid used for the transformation)
* Competent bacteria (XL1 Blue or K12)
* Plasmid (We designed and received from the Integrated DNA Technologies)

##### Protocol :

* Take the competent bacteria out of the freezer at -80°C and let thaw on ice for 20 to 30 minutes
* Take the Petri-dishes out of the fridge at 4°C and let them warm up at room temperature
* Dilute the DNA solutions in sterile water to obtain a concentration of 10ng/uL. Take 1 uL of this dilution and add it to 50uL of competent bacteria in a centrifugation tube. Mix delicately by tapping the bottom of the tube a few times
* Incubate the DNA/competent bacteria mix on ice for 30 minute
* Do a heat shock of each transformation tube by putting ½ or ⅔ of the tube in the water bath at 42°C for 30 to 60 sec
* Put the tubes back on ice for 2min
* Add 500 uL of LB (without antibiotics) to the bacteria and let grow 45 min in an agitator at 37°C . Place the tubes in a horizontal position
* Pour 50uL of the transformation on a Petri dish containing the antibiotic associated with the plasmid used during the transformation
* Incubate the Petri dishes at 37°C overnight

## All Protocols

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Nantes/Results

[![](https://static.igem.org/mediawiki/2019/thumb/e/ec/T--Nantes--logo.jpg/661px-T--Nantes--logo.jpg)](https://2019.igem.org/Team%3ANantes)

[Judging form](https://igem.org/2019_Judging_Form?team=Nantes)

Awards

[Medals](https://2019.igem.org/Team%3ANantes/Medals)

[Awards](https://2019.igem.org/Team%3ANantes/Awards)

Achievements

[Results](https://2019.igem.org/Team%3ANantes/Results)

[Model](https://2019.igem.org/Team%3ANantes/Model)

[Collaborations](https://2019.igem.org/Team%3ANantes/Collaborations)

[Parts](https://2019.igem.org/Team%3ANantes/Parts)

[Demonstrate](https://2019.igem.org/Team%3ANantes/Demonstrate)

[Improve](https://2019.igem.org/Team%3ANantes/Improve)

Human Pratices

[Integrated](https://2019.igem.org/Team%3ANantes/Human_Practices)

[Education-Engagement](https://2019.igem.org/Team%3ANantes/Public_Engagement)

Project

[Description](https://2019.igem.org/Team%3ANantes/Description)

[Design](https://2019.igem.org/Team%3ANantes/Design)

[Experiments](https://2019.igem.org/Team%3ANantes/Experiments)

[Notebook](https://2019.igem.org/Team%3ANantes/Notebook)

[Safety](https://2019.igem.org/Team%3ANantes/Safety)

[Attributions](https://2019.igem.org/Team%3ANantes/Attributions)

Team

[Team](https://2019.igem.org/Team%3ANantes/Team)

[Sponsors](https://2019.igem.org/Team%3ANantes/Sponsors)

Results
Results

# Introduction

The goal of our project Bio’Clock is to control gene expression in time by using the sugar hierarchy naturally present in E.coli (see project [description](https://2019.igem.org/Team%3ANantes/Description) and [design](https://2019.igem.org/Team%3ANantes/Design)). To begin building this tool we designed 6 different
constructs. Four simple insert constructs, each containing a sugar-specific promoter associated to a fluorescent reporter gene :

![](https://static.igem.org/mediawiki/2019/a/a0/T--Nantes--oneone.jpg)

GFP : Green Fluorescent Protein
CFP : Cyan Fluorescent Protein
RFP : Red Fluorescent Protein
YFP : Yellow Fluorescent Protein
pLAC : Lactose specific promoter
pARA : Arabinose specific promoter
pSRL : Sorbitol specific promoter
pRIB : Ribose specific promoter

Two double insert constructs, each containing 2 sugar-specific promoters, each associated with their own fluorescent reporter gene :

![](https://static.igem.org/mediawiki/2019/3/36/T--Nantes--plasmiddouble.png)

The first part of our results demonstrate how we determined if the promoters in each of these constructs functioned properly. We then explain how we established the experimental link between the concentration of sugar in the medium and the promoter activity,
before presenting how we tested the specificity of each promoter to it’s associated sugar. And finally, we present the work we did to optimize our part pARA, before finishing on the future plans for this project.

# I - Testing of the functionality of our designed parts

After transforming K12 MG1655 E.coli with our 6 different constructs, we measured by spectrofluorometry the fluorescence of the transformed bacteria cultivated in M9 medium in the presence of saturating concentration
(0.2%) of the associated sugar (for the simple insert) or the associated sugars (for the double inserts). The fluorescence studied in the cases below has been normalized with the Optical Density (OD).

## a) Simple insert constructs

We transformed K12 MG1655 E.coli with 4 different simple insert constructs :

* pLAC-GFP : plasmid containing a lactose specific promoter associated with a GFP reporter gene
* pARA-CFP : plasmid containing a arabinose specific promoter associated with a CFP reporter gene
* pSRL-RFP : plasmid containing a sorbitol specific promoter associated with an RFP reporter gene
* pRIB-YFP : plasmid containing the ribose specific promoter associated with a YFP reporter gene

![](https://static.igem.org/mediawiki/2019/a/a0/T--Nantes--oneone.jpg)

Our spectrofluorometric measurements were carried out on the transformed bacteria cultivated in a M9 medium in 2 different conditions : with 0.2% of the specific sugar, and without sugar.

#### ● pLAC-GFP: E.coli:

![](https://static.igem.org/mediawiki/2019/2/2b/T--Nantes--graph1.png)

Figure 1: GFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pLAC in the presence (green) or absence (grey) of Lactose at 0,2%. Experiments were conducted during 17 hours.

The activity of pLAC is relative to the expression of GFP fluorescence. In the condition without sugar we observe little increase in GFP fluorescence and therefore little activity of the pLAC promoter. This control shows that there is very little promoter
activity in the absence of sugar. This validates our control. In the condition where 0.2% lactose was added, we observe significant GFP levels compared to the condition without sugar starting at hour 1. The level of GFP increases linearly
over time. The reporter gene is expressed following the activation of the pLAC promoter in the presence of lactose. We observe an increase in GFP during the first 6 hours of the bacterial culture, but from the sixth hour this increase
slows down. This verifies the proper functioning of our designed pLAC promoter.

#### ● pARA-CFP: E.coli :

![](https://static.igem.org/mediawiki/2019/4/43/T--Nantes--graph2.png)

Figure 2:CFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pARA in the presence (cyan) or absence (grey) of arabinose at 0,2%. Experiments were conducted during
17 hours.

The activity of pARA is relative to the expression of CFP fluorescence. In the condition “without sugar”, there is no increase in CFP. There is therefore no promoter activity. This validates our control. In the presence of 0.2% arabinose, we observe a
strong increase in CFP expression starting in the second hour of the bacterial culture. The CFP in this condition is significantly different than the CFP in the absence of sugar. This verifies the proper functioning of our designed
pARA promoter.

#### ● pSRL-RFP E.coli :

![](https://static.igem.org/mediawiki/2019/6/6f/T--Nantes--graph3.png)

Figure 3: RFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pSRL in the presence (red) or absence (grey) of sorbitol at 0,2%. Experiments were conducted during
17 hours.

The activity of pSRL is relative to the expression of RFP fluorescence. We do not observe a significant difference of RFP fluorescence between both conditions, with or without sorbitol. Either (i) there is a RFP fluorescence in both conditions meaning
that the promoter pSRL is active even in the absence of sugar, or (ii) we are in presence of an experimental error.

#### ● pRIB-YFPE.coli :

![](https://static.igem.org/mediawiki/2019/d/db/T--Nantes--graph4.png)

Figure 4: YFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pRIB in the presence (yellow) or absence (grey) of ribose at 0,2%. Experiments were conducted during
17 hours.

The activity of pRIB is relative to the expression of YFP fluorescence. In the condition “without sugar”, there is little increase in YFP. There is therefore no significant promoter activity in the absence of sugar. This validates our control. With 0.2%
ribose, starting from the 4.5th hour, we observe a strong increase in YFP which is significantly different than the YFP observed in the condition without sugar. Therefore, we can confirm that ribose activates the expression of YFP
by increasing the activity of pRIB. This verifies the proper functioning of our designed pRIB promoter.

## b) Double insert constructs

We transformed K12 MG1655 E.coli with 2 different double insert constructs :

* “pLAC-GFP + pSRL-RFP” : plasmid containing 2 sugar specific promoters pLAC and pSRL associated with GFP and RFP reporter genes respectively
* “pARA-CFP + pRIB-RFP” : plasmid containing 2 sugar specific promoters pARA and pRIB associated with CFP and RFP reporter genes respectively

![](https://static.igem.org/mediawiki/2019/3/36/T--Nantes--plasmiddouble.png)

Our spectrofluorometric measurements were carried out on the transformed bacteria cultivated in a M9 medium in 3 different conditions : with 0.2% of the first specific sugar, with 0.2% of the second specific sugar, and without sugar.

#### ● pLAC-GFP/pSRL-RFP E.coli:

The activity of pLAC is relative to the expression of GFP fluorescence, and the activity of pSRL to RFP fluorescence.

Measurements done in the presence of 0.2% lactose and without sugar:

![](https://static.igem.org/mediawiki/2019/0/05/T--Nantes--graph5.png)

Figure 5: GFP(a) and RFP(b) fluorescence in K12 bacteria with the double insert (pLAC-GFP + pSRL-RFP), in the absence (grey) or presence of 0.2% lactose (green for GFP or red for RFP). Experiments were conducted
during 17 hours

In figure 5a, as expected, we observe a significant difference between GFP levels in the presence of lactose and without the sugar. The pLAC in our double insert construction is not activated in the absence of sugar (as previously shown for the single
insert construction, Figure 1).

Likewise, in Figure 5b, in the absence or presence of lactose there is no difference in the emission due to RFP. Lactose did not induce an increase in the pSRL promoter activity.

Therefore, pLAC is significantly activated in the presence of lactose, however the presence of lactose does not affect the activity of pSRL

Measurements done in the presence of 0.2% sorbitol and without sugar:

![](https://static.igem.org/mediawiki/2019/4/4b/T--Nantes--graph6.png)

Figure 6:GFP(a) and RFP(b) fluorescence in K12 bacteria with the double insert (pLAC-GFP + pSRL-RFP), in the absence (grey) or presence of 0.2% sorbitol (green for GFP or red for RFP). Experiments were conducted
during 17 hours

In Figure 6a we notice an increase in GFP fluorescence with 0.2% sorbitol and no increase of the GFP fluorescence in the absence of the sugar. This means that sorbitol is able to activate pLac promoter and this corroborates the data published in the paper
of Dr. Guy Aidelberg et al (1).

In figure 6b, we notice that sorbitol even in saturating concentrations (0.2%), do not activate pSrl promoter and hence RFP fluorescence in the context of a double insert with the pLac promoter. There could be two explanations for this observation. First,
it could be that our pSrl promoter is not functioning. This would explain our data in figure 6b and 3. Indeed, the promoter in the single insert construction (see above) is not activated by sorbitol

The fact that sorbitol strongly activates the pLac promoter as shown by GFP fluorescence, could also mean that sorbitol would preferentially activates pLac promoter in place of pSrl promoter. This result has never been shown before because no binary combination
of both of these promoters were tested before. Our data could suggest that there is indeed a hierarchy in the activation of promoters. A certain amount of Sorbitol will activate pLac if the latter is present. We cannot so far say if
both promoters could be activated and to which level one compared to the other because our pSrl insert does not seem to function

#### ● pARA-CFP/pRIB-RFP E.coli :

The activity of pARA is relative to the expression of CFP fluorescence, and the activity of pRIB to RFP fluorescence.

Measurements done in the presence of 0.2% arabinose and without sugar:

![](https://static.igem.org/mediawiki/2019/b/bf/T--Nantes--graph7.png)

Figure 7: CFP(a) and RFP(b) fluorescence in K12 bacteria with the double insert (pARA-CFP + pRIB-RFP), in the absence (grey) or presence of 0.2% arabinose (cyan for CFP or red for RFP). Experiments were conducted
during 17 hours.

We observe in Figure 7a an increase of CFP fluorescence in the presence of 0.2% arabinose after a latency of about 4.5h and no increase of the same fluorescence in the absence of the sugar. This validates our control (without sugar) but also indicates
that the pARA promoter in the context of the double insert is indeed activated by arabinose.

Figure 7b shows that for the first 8 hours, we do not have a significant difference in the fluorescence of RFP between the two conditions tested, i.e with or without 0.2% arabinose. After 8h, there is an increase in RFP fluorescence relative to that observed
in the absence of the sugar, indicating an increase in the pRIB promoter activity induced by the sugar. As detailed in the Dr. Guy Aidelberg et al (1) paper, arabinose can indeed very slightly activate pRIB promoter. This hence corroborates
their observations which was done with a single pRIB insert. It is noteworthy that in our case we have performed the measurements every 30 min during 17h while Aidelberg et al established activation level of pRIB by arabinose on a
single data point (at mid-exponential growth). Interestingly, the delay for the activation of pRIB by arabinose is 8h, twice the time needed for it’s activation with ribose only (see figure 4). This is an original result. Surprisingly,
we note a sharp increase in RFP fluorescence in the microplate wells with the double insert (pARA/pRIB) very early during bacterial growth from t=0h to t=2h. This increase may be artifactual due to some experimental error

Measurements done in the presence of 0.2% ribose and without sugar:

![](https://static.igem.org/mediawiki/2019/0/0a/T--Nantes--graph8.png)

Figure 8: CFP(a) and RFP(b) fluorescence in K12 bacteria with the double insert (pARA-CFP + pRIB-RFP), in the absence (grey) or presence of 0.2% ribose (cyan for CFP or red for RFP). Experiments were conducted
during 17 hours

We observe a significant linear increase of CFP in the presence of ribose when compared to the experiment without sugar, validating our control.

In the presence of ribose (Figure 8a), CFP fluorescence increases linearly throughout the experiment and has a maximum level at around 17 000 a.u obtained at hour 17. This means that ribose is able to activate the pARA promoter. This corroborates with
the experimental data published by Guy Aidelberg et al (1).

When pRIB activity is monitored in the presence or absence of 0.2% ribose in the context of a double insert with pARA, there is a difference that is observed between the RFP fluorescence as from 4.5h. This confirms that pRIB promoter is indeed activated
by ribose. Interestingly seems to be an immediate activation of pARA by ribose while there is a delay in the activation of pRIB by the same and similar to what was observed in figure 7b (activation of pRIB with arabinose that exhibited
a delay of 4.5h).

## Summary

In the simple insert constructs, we conclude that 3 out of 4 of our parts (pLAC, pARA and pRIB) are significantly activated in the presence of their associated sugar compared to conditions without sugar, verifying the proper functioning of these parts.
Unfortunately, we were not able to have pSRL construct to work properly.
 In the case of the double insert constructs, we observed an activation of pLAC, pARA and pRIB in the presence of their respective sugars.

 They are all activated but to a lesser degree in the presence of the other sugar, thus confirming the existence of cross-activation and a sugar hierarchy in E.coli.

# II - The link between sugar concentration and promoter activity

For these tests we measured the fluorescence in K12 MG1655 E.coli transformed with each of our 6 constructs. The bacteria were cultivated in M9 medium with various different concentrations of the associated sugar (for simple insert constructs),
or associated sugars (for double insert constructs). The fluorescence studied in the graphs below has been normalised with the Optical Density (OD).

## a) Simple insert constructs

As a reminder, in our 4 simple insert constructs :

* pLAC is associated to GFP
* pARA is associated to CFP
* pSRL is associated to RFP
* pRIB is associated to YFP

#### ● pLAC-GFP E.coli :

![](https://static.igem.org/mediawiki/2019/e/e8/T--Nantes--graph9.png)

Figure 9: GFP Fluorescence normalised with OD for E.coli K12 MG1655 transformed with pLAC in selected concentrations of Lactose

The activity of pLAC is relative to the expression of GFP fluorescence. We observe a slight increase of GFP fluorescence in the condition without sugar, but the fluorescence level remains relatively insignificant when compared to the fluorescence in conditions
where lactose is present. However we do not observe a significant difference of GFP expression between the different conditions containing lactose. The concentration of lactose does not seem to have a significant effect on the activity
of our designed pLAC in the simple insert construct. However for this conclusion to be decisive we would need to carry out more experiments with different sugar concentrations

#### ● pARA-CFP E.coli :

![](https://static.igem.org/mediawiki/2019/b/b3/T--Nantes--graph10.png)

Figure 10:CFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pARA in selected concentrations of arabinose

The activity of pARA is relative to the expression of CFP fluorescence.
 Without sugar, we observe a very low level of CFP fluorescence which allows us to validate the proper functioning of our pARA promoter in this condition. The
difference of CFP expression between conditions with arabinose and the condition without sugar becomes significant around the fourth hour of bacterial culture.
 From 14.5h, we notice a significant difference of CFP expression amongst
the conditions containing 0.1 %, 0.05% and 0.01% of arabinose. However, the curve of the condition containing 0.02% of arabinose follows the same tendency as the previous curves, but it has enormous error bars. If we consider the results
of this condition to be distorted, we can conclude that the intensity of the activation of pARA can be regulated proportionally to the concentration of arabinose in the medium

#### ● pSRL-RFP E.coli :

![](https://static.igem.org/mediawiki/2019/d/dd/T--Nantes--graph11.png)

Figure 11: RFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pSRL in selected concentrations of Sorbitol

The activity of pSRL is relative to the expression of RFP fluorescence. This figure shows no significant difference of RFP expression between all the different conditions This is coherent with our previous observations whereby we observed that pSRL activity
was equivalent in absence and in presence of 0.2% sorbitol (see figure 3). The pSRL does not seem to be affected by the sugar in the medium, therefore we assume a dysfunctionality of this promoter in our constructs

#### ● pRIB-YFP E.coli :

![](https://static.igem.org/mediawiki/2019/4/42/T--Nantes--graph12.png)

Figure 12: YFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pRIB in selected concentrations of Ribose.

The activity of pRIB is relative to the expression of YFP fluorescence.

In the condition without sugar, we observe a very low, stable and significantly different YFP fluorescence compared to the YFP in the other conditions (YFP max is around 10 000 a.u compared to 40 000 and 70 000 for the other conditions).

In the conditions with sugar, we notice the same curve tendencies with an increase in YFP fluorescence around the third hour, however there is no significant difference of YFP levels between these different conditions.

We do notice one anomaly for the condition 0.01% ribose. At hour 12, the YFP fluorescence strongly increases (from around 40 000 au to 70 000 a.u.) during the last 4 hours of the culture.

We suspect either a measurement anomaly on these plate wells, an error during the preparation of the medium for this condition, or an unidentified phenomenon (this experience was repeated twice and the same curve was observed both
times).

The promoter pRIB was indeed activated in the presence of ribose during this experiment, however, does not seem sensitive to the change of concentration of this sugar. The intensity of the activity of pRIB in our construct does
not seem to depend on the concentration in ribose present in the medium.

## b) Double insert constructs

As a reminder, in our 2 double insert constructs :

* pLAC-GFP-pSRL-RFP ; pLAC is associated to GFP, and pSRL to RFP
* pARA-CFP-pRIB-YFP ; pARA is associated to CFP, and pRIB to RFP

#### ● pLAC-GFP/pSRL-RFP E.coli :

The activity of pLAC is relative to the expression of GFP fluorescence, and the activity of pSRL to RFP fluorescence.

Measurements done in the presence of different concentrations of lactose and without sugar:

![](https://static.igem.org/mediawiki/2019/a/a7/T--Nantes--graph13.png)

Figure 13:GFP(a) and RFP(b) normalized with OD for E.coli K12 MG1655 transformed with pLAC/pSRL in selected concentrations of Lactose

Without sugar, the GFP fluorescence does not significantly change (very slight increase from 400 au to 500 a.u.), and remains significantly inferior to the GFP fluorescence in presence of lactose.

The GFP signal is lower with 0.01% lactose in comparison to the signals observed with higher concentrations of the sugar (figure 13a). There is no significant difference in GFP fluorescence levels between experiments with lactose
concentrations at 0.02%, 0.05% and 0.1%. Maximal activation of pLAC promoter seems to be reached as from 0.02% lactose.

The RFP fluorescence does not seem to have any increase over time, or to have any significant difference between the different conditions. Therefore, there is no significant pSRL activity due to the presence of lactose. It might
be because the pSRL promoter is not functional as it was seen in Figures 3, 5b and 6b. So we can conclude that we were not successful to have a function pSRL promoter.

![](https://static.igem.org/mediawiki/2019/c/c6/T--Nantes--graph14.png)

Figure 14:(a)GFP and (b)RFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pLAC/pSRL in selected concentrations of Sorbitol

In Figure 14a we observe that the GFP without sugar and with 0.01% sorbitol are not significantly different. 0.01% sorbitol is not enough to activate pLAC promoter. Significant activation of this promoter is only observed after 6 hours for concentration of
sorbitol of 0.02% or above and would be concentration dependent. Though maximal activation is reached with 0.05% sorbitol

In figure 14b we observe no significant difference between any of the sorbitol concentrations. As mentioned above for Figure 13b, our results seem to indicate that pSRL promoter is unfortunately not functional

#### ● pARA-CFP/pRIB-RFP E.coli :

The activity of pARA is relative to the expression of CFP fluorescence, and the activity of pRIB to RFP fluorescence.

Measurements done in the presence of different concentrations of arabinose and without sugar:

![](https://static.igem.org/mediawiki/2019/c/c2/T--Nantes--graph15.png)

Figure 15: CFP (a) and RFP (b) Fluorescence normalized with OD for  K12 MG1655 transformed with pARA/pRIB in selected concentrations of Arabinose

We observe in Figure 7a an increase if CFP fluorescence in the presence of 0.2% arabinose after a latency of about 4.5h and no increase of the same fluorescence in the absence of the sugar. This validates our control (without sugar) but also indicates
that the pARA promoter in the context of the double insert is indeed activated by arabinose.

In the conditions without sugar for both figures, we observe a stable fluorescence. Both promoters do not seem to be significantly activated in the absence of arabinose.

 In both graphs, the fluorescence in the presence of arabinose become significantly different from the fluorescence in the absence of sugar from around hours 7 and 8 of the culture.

 Our data do not show a concentration dependant variation of GFP or RFP fluorescence. Both pARA and pRIB promoters are very active at low concentrations of arabinose. This is an original result

Measurements done in the presence of different concentrations of ribose and without sugar:
![](https://static.igem.org/mediawiki/2019/7/78/T--Nantes--graph16.png)

Figure 16: CFP (a) and RFP (b) Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pARA/pRIB in selected concentrations of Ribose

We observe a significant linear increase of CFP in the presence of ribose when compared to the experiment without sugar, validating our control.

In figure 16a, CFP fluorescence seems not to vary significantly between any of the conditions. There does not seem to be any particular activity of the pARA promoter in the presence of ribose in the medium.

 In figure 16b, there is a non significant variation in RFP fluorescence in the absence of ribose. In presence of the sugar, the RFP fluorescence is increasing significantly after 6.5-7h. The increase is concentration dependant.

 This experimental setup where the two promoters were combined and tested against ribose is original. Here the highest concentration of ribose used was 0.1%. It seems to be insufficient to activate the pARA promoter while it
was able to do it at a concentration of 0.2%. We can hypothesize that in the context of this double insert, ribose preferentially activates its own promoter pRIB and not pARA, when it is at a concentration of 0.1% or below. At
higher concentrations (like at 0.2%), the pRIB promoter is saturated and some ribose is available for activating pARA (Figure 8).

## Summary

In our simple insert constructs, we showed that pARA is the only promoter that responded to the concentration level of its cognate sugar. For two other promoters, pLAC and pRIB, a high level of activation was rapidly reached with a low concentration of
their cognate sugars. We were not successful to demonstrate that our pSRL construct was functioning correctly. In our double constructs, pLAC/pSRL and pARA/pRIB, our results corroborates with known hierarchy of the sugars. Sorbitol indeed was able
to activate pLAC promoter with concentrations higher than 0.02%. Arabinose is able to cross-activate pRIB and reciprocally, ribose is able to activate pARA but only at high concentrations and will preferentially activate its cognate promoter.

# III - The specificity of each promoter to their associated sugar

To complete our experiments we tested the promoter activity of the simple insert constructs pLAC-GFP and pRIB-YFP, in K12 MG1655 E.coli, in the presence of saturation concentration of different sugars. Due to a lack of time, we were unable to
carry out these experiments on our other constructs pARA and pSRL. The primary purpose of these measurements is to confirm already published cross-activation results.

#### ● pLAC-GFP E.coli :

![](https://static.igem.org/mediawiki/2019/f/fd/T--Nantes--graph17.png)

Figure 17: GFP fluorescence normalized with the OD in E.coli K12 MG1655 bacteria transformed with pLAC-GFP construct in the presence of saturating amounts of different sugars

The activity of pLAC is relative to the expression of GFP fluorescence.

 In absence of any sugar, we observe no increase of GFP fluorescence during the 15 hours of culture . This validates our negative control.

On the other hand, all tested sugars were able to induce an increase of GFP fluorescence hence activate the pLAC promoter. This corroborates already published results. At this stage we cannot conclude on the differences between the
sugars regarding the fluorescence levels reached after 5, 10 or 15 hours since we did not quantify the amount of proteins and only normalized the data with bacterial growth OD values.

#### ● pRIB-YFP E.coli :

![](https://static.igem.org/mediawiki/2019/0/04/T--Nantes--graph18.png)

Figure 18: YFP fluorescence normalized with the OD in E.coli K12 MG1655 bacteria transformed with pRIB-YFP construct in the presence of saturating amounts of different sugars.

The activity of pRIB is relative to the expression of YFP fluorescence.

 In the condition without sugar we notice an increase in YFP over the first 5 hours which then stagnates after hour 5. This is similar to what was observed in Figures 7b and 8b where without sugar, there is a slight but sharp increase
of RFP fluorescence corresponding to pRIB activity over the first 5 hours before stabilization of the fluorescence. We can hypothesize that this increase could be due to internal ribose produced by the bacterie and that enters in the composition
of its DNA or RNA.

 Only three sugars, arabinose, sorbitol and ribose were able to induce an YFP fluorescence higher than the condition without sugar. Maximum activation of pRIB is reached with its cognate sugar ribose. On the other hand, activation
of pRIB by lactose follow a very similar pattern as the negative control (in the absence of sugar). Hence, lactose does not seem to induce pRIB promoter activity. This last observation corroborates already published data by Guy Aidelberg et al (1).

## Summary

pLAC seems to be activated by all 4 sugars. pRIB is only activated by 3 sugars (arabinose, sorbitol and ribose) and is not activated by lactose. These confirm cross-activation data published previously by Guy Aidelberg et al (1).
 We hypothesize
that internal bacterial ribose produced for DNA and RNA may explain the sudden and slight activation of pRIB promoter in the absence of any added sugar in the medium.

# IV - The optimisation of pARA

One of our goals was to improve a part present in the iGEM “part registry”. In doing so, we wanted to continue working on sugar activated promoters. Therefore we chose 2 different promoters associated with the consumption of arabinose.

* part:BBA I13453 → that we call “1” from this point
* part:BBA K206000 → that we will call “3” from this point

We ordered these parts associated with a CFP reporter gene, as well as a second version of these parts with the consensus sequence of
[CRP](https://2019.igem.org/Team%3ANantes/Model) (AAATGTGATCTAGATCACATT).

After transforming top10 E.coli we obtained 4 different strains to be tested :

* “1” : pIDT pARAI13453 - CFP
* “2” : pIDT pARAI13453 optimized - CFP
* “3” : pIDT pARAK206000 - CFP
* “4” : pIDT pARAK206000 optimized -CFP
* “5” : pIDT pARAI13453 - GFP
* “6” : pIDT pARAI13453 optimized - GFP

 We then cultivated our bacteria in M9 medium with 0.2% arabinose. Our 4 different bacterial cultures were followed by spectrofluorometry for 17 hourss. The fluorescence observed in the graphs below has been normalized with the Optical
Density (OD).

#### ● Top10 E.coli 1 and 2

As a reminder, in this case the top10 E.coli was transformed with plasmids containing the pARA part I13453 either optimized with the consensus sequence of CRP (AAATGTGATCTAGATCACATT) (“2”), and non optimized (“1”).

![](https://static.igem.org/mediawiki/2019/4/4e/T--Nantes--graph19.png)

Figure 19: CFP Fluorescence normalized with OD for E.coli T10 transformed with the part 1 and 2 in absence of any sugar (◇), in the presence of 0.2% arabinose (△) , 0,1% arabinose (☐), 0,05 % arabinose (+), 0.02% arabinose
(○) and M9 rich medium (x)

Both strains express CFP, but we observe no difference between the optimised pARA part CFP (2) and the non optimised part (1). We can conclude that the optimisation by addition of the consensus sequence of CRP (AAATGTGATCTAGATCACATT) does not seems to have significant effect on the activity of the pARA I13453.

#### ● Top10 E.coli3 and 4

As a reminder, in this case the top10 E.coli was transformed with plasmids containing the pARA part K206000 either optimized with the consensus sequence of CRP (AAATGTGATCTAGATCACATT) (“4”), and non optimized (“3”).

![](https://static.igem.org/mediawiki/2019/9/94/T--Nantes--graph20.png)

Figure 20: CFP Fluorescence normalized with OD for E.coli T10 transformed with the part 3 and 4 in absence of any sugar (◇), in the presence of 0.2% arabinose (△) , 0,1% arabinose (☐), 0,05 % arabinose (+), 0.02% arabinose
(○) and M9 rich medium (x)

Both strains express CFP, but we observe no difference between the optimised pARA part CFP (3) and the non optimised part (4). We can conclude that the optimisation by addition of the consensus sequence of CRP (AAATGTGATCTAGATCACATT) does not seems to have significant effect on the activity
of this promoter too.

#### ● Top10 E.coli 5 and 6

As a reminder, in this case the top10 E.coli was transformed with plasmids containing the pARA part I13453 either optimized with the consensus sequence of CRP (AAATGTGATCTAGATCACATT) (“5”), and non optimized (“6”).

![](https://static.igem.org/mediawiki/2019/7/7d/T--Nantes--graph21.png)

Figure 21: GFP Fluorescence normalized with OD for E.coli T10 transformed with the part 5 and 6 in the absence of any sugar (◇), in the presence of 0.2% arabinose (△) , 0,1% arabinose (☐), 0,05 % arabinose (+), 0.02%
arabinose (○) and M9 rich medium (x).

Both strains express GFP, but we observe no difference between the optimised pARA part GFP (5) and the non optimised part (6). We can conclude that the optimisation by addition of the consensus sequence of CRP (AAATGTGATCTAGATCACATT) seems to not have a significant effect on the activity.

## Summary

Here we have shown that the consensus sequence of CRP (AAATGTGATCTAGATCACATT) seems to not have changed the level of expression of our different pARA promoters. However, it seems that the M9 rich medium have an impact on the activation level of pARA. We do
not have an explanation to why the M9 rich medium seems to enhance the activation of our promoters, an experimental error may be possible.

# V - Binary combinations of sugars with simple and double inserts

Here we tested the activities of single or double inserts with binary combinations of sugars. The tested single inserts are pLAC-GFP, pARA-CFP and pRIB-YFP. The only tested double insert was pARA-CFP/pRIB-RFP. The primary purpose of these experiments is to analyse the activity of each promoter in the presence of 2 sugars at a time: it’s own specific sugar and another. We will see if the sugar-hierarchy is still present in our constructions in these conditions, and how a sugar,higher or lower in the hierarchy impacts the expression of the different promoters.

#### ● pLAC-GFP E.coli :

As a reminder, here E.coli K12 MG1655 was transformed with pLAC-GFP. We monitored pLAC activity by measuring GFP fluorescence in absence or presence of combinations of sugars. Results are provided below (Fig. 22) .

![](https://static.igem.org/mediawiki/2019/d/d3/T--Nantes--graph22.png)

Figure 22: Figure 22. GFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pLAC-GFP in absence of any sugar (■), in presence of 0.2% lactose combined with M9 rich medium (●), 0.2% arabinose (○),
0.2% sorbitol (△) and 0.2% ribose (+). Experiments were conducted during 17 hours

Our results show that pLAC promoter is activated by all tested binary combinations of sugars which all contained 0.2% lactose. The increase in activity over time is mainly imputable to lactose. Addition of other sugars known to be lower in the
hierarchy with respect to lactose did not induced an additional effect on pLAC activity. This confirms that pLAC is on the top of the hierarchy

#### ● pARA-CFP E.coli :

As a reminder, here E.coli K12 MG1655 was transformed with pARA-CFP. We monitored pARA activity by measuring CFP fluorescence in absence or presence of combinations of sugars. Results are provided below (Fig. 23)

![](https://static.igem.org/mediawiki/2019/6/6f/T--Nantes--graph23.png)

Figure 23: CFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pARA-CFP in absence of any sugar (■), in the presence of 0.2% arabinose combined with M9 rich medium (●), 0.2% lactose (○), 0.2% sorbitol
(△) and 0.2% ribose (+). Experiments were conducted during 17 hours.

No change in CFP fluorescence is observed in the absence of sugar. This validates our negative control.

Contrasting results were obtained for the different combinations of sugars. In presence of saturating arabinose (0.2%) and M2 rich medium, CFP fluorescence slightly increased over time with a fluorescence level that was slightly above
that of the negative control after 17 hours. A similar pattern is observed when 0.2% arabinose is combined with 0.2% lactose. This would suggest that M9 rich medium likewise lactose would have an inhibitory effect on pARA activity. Indeed,
when arabinose is combined with others sugars lower in the hierarchy, i.e sorbitol or ribose, the activity of pARA promoter was much higher. With sorbitol, the CFP fluorescence increased steadily between 5 hours and 10 hours of culture to
reach a plateau after the 10th hour. When combined with 0.2% ribose, the fluorescence pattern was different as it exhibited a steady linear increase over time.

These results show that ribose and sorbitol have a cumulative effect with arabinose that leads to a bigger activation of the pARA promoter.

#### ● pRIB-YFP E.coli :

As a reminder, here E.coli K12 MG1655 was transformed with pRIB-YFP. We monitored pRIB activity by measuring YFP fluorescence in absence or presence of combinations of sugars. Results are provided below (Fig. 24).

![](https://static.igem.org/mediawiki/2019/b/bc/T--Nantes--graph24.png)

Figure 24: YFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pRIB-YFP in absence of any sugar (■), in the presence of 0.2% ribose combined with M9 rich medium (●), 0.2% lactose (○), 0.2% arabinose
(△) and 0.2% sorbitol (+). Experiments were conducted during 17 hours.

Here, for all tested conditions, there is a sharp increase in YFP fluorescence during the first 2.5 hours. This is similar to our previous results as shown in Figures 7b, 8b and 18. As mentioned above, we hypothesize that this could result from endogenous
ribose produced by the bacteria for DNA and RNA that could activate pRIB promoter.

When 0.2% ribose was combined with M9 rich medium or 0.2% lactose, no additional increase of YFP fluorescence was observed when compared to the fluorescence in absence of sugar, indicating that these may strongly inhibit the action of
ribose on pRIB promoter. On the other hand, sorbitol and arabinose when combined with ribose did not inhibit pRIB promoter activity. Combined with arabinose, YFP fluorescence levels reached slightly higher levels than when combined with sorbitol.

#### ● pARA-CFP/pRIB-RFP E.coli :

As a reminder, here E.coli K12 MG1655 was transformed with a double insert containing pARA-CFP/pRIB-RFP. We monitored both pARA and pRIB activities by measuring CFP and RFP fluorescence respectively in absence or presence of combinations of sugars.
Results are provided below (Fig. 25).

![](https://static.igem.org/mediawiki/2019/2/22/T--Nantes--graph25.png)

Figure 25: CFP Fluorescence normalized with OD for K12 MG1655 transformed with pARA-CFP/pRIB-RFP double insert in absence of any sugar (■), in the presence of 0.2% arabinose + 0.2% ribose (●), 0.2% arabinose + 0.2% lactose
(○), 0.2% arabinose + 0.2% sorbitol (△), 0.2% ribose + 0.2% lactose (+) and 0.2% ribose + 0.2% sorbitol (◇). Experiments were conducted during 17 hours.

Results in figure 25 show that in absence of sugar, there is no significant change in CFP fluorescence. This validates the negative control for CFP fluorescence.

In presence of 0.2% arabinose combined with 0.2% lactose or 0.2% ribose, CFP fluorescence that is supposed to monitor pARA activity showed a steady increase as from 6 hours of culture. This suggests that neither lactose nor ribose inhibits
pARA promoter activity.

Similarly, when 0.2% arabinose was combined with 0.2% sorbitol, a steady increase in pARA activity as from the 7.5th hour was also observed but with much lower levels of CFP fluorescence reached when compared to the combination with lactose
or ribose.

The combination of ribose with lactose also induced a steady increase in CFP fluorescence as from 7.5 hours that reaches a level that is roughly 50% to that reached when arabinose was combined with lactose or ribose. Similarly, when 0.2% ribose was combined
with 0.2% sorbitol, a steady increase in pARA activity as from the 7.5th hour was also observed but with much lower levels of CFP fluorescence as for arabinose + sorbitol.

![](https://static.igem.org/mediawiki/2019/d/da/T--Nantes--graph26.png)

Figure 26: RFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pARA-CFP/pRIB-RFP double insert in absence of any sugar (■), in the presence of 0.2% arabinose + 0.2% ribose (●), 0.2% arabinose +
0.2% lactose (○), 0.2% arabinose + 0.2% sorbitol (△), 0.2% ribose + 0.2% lactose (+) and 0.2% ribose + 0.2% sorbitol lactose (◇). Experiments were conducted during 17 hours.

We also monitored the RFP fluorescence when the double insert construct was tested with combinations of sugars (Figure 26).

The results show that pRIB activity measured as RFP fluorescence was not significantly changed in all tested conditions. We can hypothesized that we did not measured any pRIB activity probably due to the fact that the sugars would either
have preferentially promoted pARA activity (e.g ribose or lactose) or inhibited pRIB activity.

## Summary We can conclude that in the simple constructs, a sugar higher up in the hierarchy will inhibit promoters associated to sugars lower in the hierarchy. For example, when pARA is placed in the presence of 0,2% arabinose and 0.2% lactose, pARA is not activated. Lactose, being higher up in the hierarchy than arabinose, inhibits pARA. A sugar which is lower in the hierarchy has no effect on the activity of a promoter associated with a sugar of higher hierarchy. For the double construct, we notice that every combination of sugar in a lower hierarchy than the arabinose produced a CFP fluorescence which results of the induction a pARA activity. It does not modified the activation of pARA. The combination of 0,2% of lactose and 0,2% of Arabinose shows an activation of pARA. It means that the Lactose did not inhibit the pARA, as we expected. It can be a cross-activation because theses sugars are close in the hierarchy.When we study the RFP fluorescence (showing the activity of pRIB), we can’t observe any significant results because every sugar are higher than Ribose in the hierarchy. All sugars will induce an inhibition of the pRIB. This results were expected.

# VI - Duration of the promoter expression

As our tool is mainly based on controlling gene expression in time, it is important for us to study the activity of these promoters in time. This will allow us to better understand the behavior of our promoters, enabling us to take into account any time differences we might discover in the design of our tool.

#### ● pLAC-GFP E.coli :

![](https://static.igem.org/mediawiki/2019/9/90/T--Nantes--graph27.jpg)

Figure 27: Rate of GFP Fluorescence/hours for E.coli K12 MG1655 transformed with pLAC-GFP simple insert in the absence or in the presence of 0,2% of Lactose

The activity of pLAC is relative to the expression of GFP fluorescence.
We do not observe the production of GFP fluorescence in the condition without sugar. It confirms our control. With 0,2% of lactose, we can observe a linear increase of GFP production per hour that begins around hour 2,5. At the 5th hour We observe a peak of production, and then the production of GFP sharply decreases until the 7th hour.
We can conclude that the activity of the promoter pLAC, observed here as the rate of GFP production, starts 2,5 hours after the presence of sugar in the medium and it’s activity lasts around 4,5 hours.

#### ● pARA-CFP E.coli :

![](https://static.igem.org/mediawiki/2019/e/ec/T--Nantes--graph28.jpg)

Figure 28:Rate of CFP Fluorescence/hours for E.coli K12 MG1655 transformed with pARA-CFP simple insert in the absence or in the presence of 0,2% of Arabinose

The activity of pARA is relative to the expression of CFP fluorescence.
We do not observe an increase in the rate of production of CFP fluorescence in the condition without sugar. It confirms our control. With 0,2% of Arabinose, we observe a steady increase of the CFP production rate beginning at hour 1,5. At hour 4,5 the rate of CFP production reaches a plateau and remains in this state throughout the end of the experiment.

We can conclude that the activity of the promoter pARA starts around 4,5 hours after the presence of sugar, and the activation remains constant.

#### ● pRIB-YFP E.coli :

![](https://static.igem.org/mediawiki/2019/9/98/T--Nantes--graph29.jpg)

Figure 29: Rate of YFP Fluorescence/hours for E.coli K12 MG1655 transformed with pRIB-YFP simple insert in the absence or in the presence of 0,2% of Ribose

The activity of pRIB is relative to the expression of YFP fluorescence.
We do not observe an increase in theYFP production rate in the condition without sugar. It confirms our control. With 0,2% of Ribose, we can observe a linear increase of YFP production rate starting at the third hour. We have a peak in the YFP production rate at the 6th hour, and then the speed of YFP production declines until the 10,5th hour.
We can conclude that the activity of the promoter pRIB starts 3 hours after the presence of sugar in the medium and it is activated during 7,5 hours.

## Summary

We can conclude that the activation and the activity of promoters vary in time, from one to the other. In saturated sugar conditions, pLAC is activated around hour 2,5 after the addition of sugar and it’s activity lasts for around 4,5 hours, pARA is activated around hour 4,5 after the addition of sugar, and the activity withheld throughout the end of the experiment, and finally pRIB was activated around hour 3 and lasted around 7,5 hours.
This information would need to be taken into account to increase the precision of sequential gene expression in time of our tool.

(1)“Hierarchy of non-glucose sugars in Escherichia Coli.” Aidelberg Guy et al, BMC Systems Biology., 2014 , PMID: 25539838