iGEM 2013 — Team Evry

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# Team:Evry/Notebook

### From 2013.igem.org

Iron coli project

[![ban provisory](http://2013.igem.org/wiki/images/8/84/EvryOfficialBanner.png)](http://2013.igem.org/Team%3AEvry)

* [![](http://2013.igem.org/wiki/images/8/8c/EvryHome.png)Home](http://2013.igem.org/Team%3AEvry)
* [![](http://2013.igem.org/wiki/images/4/4f/EvryProject.png)Project](http://2013.igem.org/Team%3AEvry/Project "Overview")
  + [Iron metabolism](http://2013.igem.org/Team%3AEvry/Project_metabolism)
  + [Related diseases](http://2013.igem.org/Team%3AEvry/Project_diseases)
  + [Iron chelators](http://2013.igem.org/Team%3AEvry/Project_FUR)
  + [Achievements](http://2013.igem.org/Team%3AEvry/Achievements)
  + [References](http://2013.igem.org/Team%3AEvry/References)
* [![](http://2013.igem.org/wiki/images/a/ad/EvryTeam.png)Team](http://2013.igem.org/Team%3AEvry/Team "Our team")
  + [iGEM Profile](http://igem.org/Team.cgi?id=1163)
  + [Attributions](http://2013.igem.org/Team%3AEvry/Attributions)
  + [Collaborations](http://2013.igem.org/Team%3AEvry/Collaborations)
  + [Acknowledgement](http://2013.igem.org/Team%3AEvry/Acknowledgement)
  + [Press](http://2013.igem.org/Team%3AEvry/Press)
  + [Photo gallery](http://2013.igem.org/Team%3AEvry/Gallery)
* [![](http://2013.igem.org/wiki/images/e/e5/EvryModel.png)Model](http://2013.igem.org/Team%3AEvry/Modeling "Overview")
  + [Flush treatment](http://2013.igem.org/Team%3AEvry/flush_model)
  + [Enterobactin production](http://2013.igem.org/Team%3AEvry/ent_prod)
  + [Genome scale](http://2013.igem.org/Team%3AEvry/Metabolism_model)
  + [Population scale](http://2013.igem.org/Team%3AEvry/pop_scale)
  + [Logistic functions](http://2013.igem.org/Team%3AEvry/LogisticFunctions)
  + [Chemical reasoning](http://2013.igem.org/Team%3AEvry/ChemicalTools)
  + [Plate Reader](http://2013.igem.org/Team%3AEvry/data_analysis)
* [![](http://2013.igem.org/wiki/images/9/90/EvryBiology.png)Biology](http://2013.igem.org/Team%3AEvry/Biology "Overview")
  + [Parts](http://2013.igem.org/Team%3AEvry/Parts)
  + [Sensor](http://2013.igem.org/Team%3AEvry/Sensor)
  + [Inverter](http://2013.igem.org/Team%3AEvry/Inverter)
  + [Chelator](http://2013.igem.org/Team%3AEvry/Chelator)
  + [Results](http://2013.igem.org/Team%3AEvry/Results)
* [![](http://2013.igem.org/wiki/images/f/fa/EvryHP.png)Human Practice](http://2013.igem.org/Team%3AEvry/HumanPractice "Overview")
  + [Consultation](http://2013.igem.org/Team%3AEvry/HumanPractice/Expert_consultation)
  + [Survey](http://2013.igem.org/Team%3AEvry/HumanPractice/Patients_perceptions)
  + [Seminar](http://2013.igem.org/Team%3AEvry/Seminar)
  + [Philosophy](http://2013.igem.org/Team%3AEvry/Philosophy)
  + [Safety](http://2013.igem.org/Team%3AEvry/Safety)
  + [Economy](http://2013.igem.org/Team%3AEvry/Economy)
  + [Art](http://2013.igem.org/Team%3AEvry/Art)
* [![](http://2013.igem.org/wiki/images/4/4b/Logo_pill.png)Capsule](http://2013.igem.org/Team%3AEvry/Pill_design)
* [![](http://2013.igem.org/wiki/images/6/6a/EvryNotebook.png)Notebook](http://2013.igem.org/Team%3AEvry/Notebook "Notebook")
  + [Protocols](http://2013.igem.org/Team%3AEvry/Protocols)
  + [Primers](http://2013.igem.org/Team%3AEvry/Primers)
* [![](http://2013.igem.org/wiki/images/3/3f/EvrySponsors.png)Sponsors](http://2013.igem.org/Team%3AEvry/Sponsors)
[![](http://2013.igem.org/wiki/images/4/47/IGEM2013logoEvry.png)](http://2013.igem.org "Go to iGEM site")

* [Protocols: BioBricks' Conception](http://2013.igem.org/Team%3AEvry/Protocols#Protocol's_overview)

* [Protocols' overview](http://2013.igem.org/Team%3AEvry/Protocols#Protocol's_overview "Protocol's overview")
* [Solutions and Media](http://2013.igem.org/Team%3AEvry/Protocols#Media "Solutions and Media")
* [DNA genomic extraction](http://2013.igem.org/Team%3AEvry/Protocols#DNA_genomic_extraction "DNA genomic extraction")
* [PCR](http://2013.igem.org/Team%3AEvry/Protocols#Polymerase_Chain_Reaction "PCR")
* [Golden Gate](http://2013.igem.org/Team%3AEvry/Protocols#Golden_Gate "Golden Gate")
* [Competent cells](http://2013.igem.org/Team%3AEvry/Protocols#Competent_cells "Competent cells")
* [Transformation](http://2013.igem.org/Team%3AEvry/Protocols#Transformation "Transformation")
* [Plasmid purification](http://2013.igem.org/Team%3AEvry/Protocols#Plasmid_purification "Plasmid purification ")
* [Ectrophoresis](http://2013.igem.org/Team%3AEvry/Protocols#Electrophoresis "Ectrophoresis")
* [PCR Purification](http://2013.igem.org/Team%3AEvry/Protocols#PCR_Purification "PCR Purification ")
* [BioBrick Assembly](http://2013.igem.org/Team%3AEvry/Protocols#BioBrick_Assembly "BioBrick Assembly ")

* [Protocols: BioBricks' Characterization](http://2013.igem.org/Team%3AEvry/Protocols#Characterization_overview)

* [Protocols' overview](http://2013.igem.org/Team%3AEvry/Protocols#Characterization_overview "Protocols' overview")
* [TECAN Analysis](http://2013.igem.org/Team%3AEvry/Protocols#TECAN_Analysis "TECAN Analysis")
* [Microfluidic experiment](http://2013.igem.org/Team%3AEvry/Protocols#Microfluidic_experiment  "Microfluidic experiment ")
* [Homologous recombination](http://2013.igem.org/Team%3AEvry/Protocols#Homologous_recombination  "Homologous recombination ")
* [Siderophore detection](http://2013.igem.org/Team%3AEvry/Protocols#Siderophore_detection  "Siderophore detection ")

* [Primers](http://2013.igem.org/Team%3AEvry/Primers)

* [JUNE](http://2013.igem.org/Team%3AEvry/Notebook/#week1)

* [Week 1](http://2013.igem.org/Team%3AEvry/Notebook#week1 "17th June - 23rd June")
* [Week 2](http://2013.igem.org/Team%3AEvry/Notebook#week2 "24th June - 30th June")

* [JULY](http://2013.igem.org/Team%3AEvry/Notebook/#week3)

* [Week 3](http://2013.igem.org/Team%3AEvry/Notebook#week3 "1st July - 7th July")
* [Week 4](http://2013.igem.org/Team%3AEvry/Notebook#week4 "8th July - 14th July")
* [Week 5](http://2013.igem.org/Team%3AEvry/Notebook#week5 "15th July - 21st July")
* [Week 6](http://2013.igem.org/Team%3AEvry/Notebook#week6 "22nd July - 28th July")

* [AUGUST](http://2013.igem.org/Team%3AEvry/Notebook#/week7)

* [Week 7](http://2013.igem.org/Team%3AEvry/Notebook#week7 "29th July - 4th August")
* [Week 8](http://2013.igem.org/Team%3AEvry/Notebook#week8 "5th August - 11th August")
* [Week 9](http://2013.igem.org/Team%3AEvry/Notebook#week9 "12th August - 18th August")
* [Week 10](http://2013.igem.org/Team%3AEvry/Notebook#week10 "19th August - 25th August")
* [Week 11](http://2013.igem.org/Team%3AEvry/Notebook#week11 "26th August - 1st September")

* [SEPTEMBER](http://2013.igem.org/Team%3AEvry/Notebook/#week12)

* [Week 12](http://2013.igem.org/Team%3AEvry/Notebook#week12 "2nd September - 8th September")
* [Week 13](http://2013.igem.org/Team%3AEvry/Notebook#week13 "9th September - 15th September")
* [Week 14](http://2013.igem.org/Team%3AEvry/Notebook#week14 "16th September - 22nd September")
* [Week 15](http://2013.igem.org/Team%3AEvry/Notebook#week15 "23rd September - 29th September")

* [OCTOBER](http://2013.igem.org/Team%3AEvry/Notebook/#week16)

* [Week 16](http://2013.igem.org/Team%3AEvry/Notebook#week16 "30th September - 6th October")
* [Week 17](http://2013.igem.org/Team%3AEvry/Notebook#week17 "7th October - 13th October")
* [Week 18](http://2013.igem.org/Team%3AEvry/Notebook#week18 "14th October - 20th October")
* [Week 19](http://2013.igem.org/Team%3AEvry/Notebook#week19 "21st October - 27th October")
* [Week 20](http://2013.igem.org/Team%3AEvry/Notebook#week20 "28th October - 3rd November")

# Week 1: 17th June - 23rd June

## Tuesday, 19th June

As a start for the labwork, we first decided to make competent cells. We grew from glycerol samples three E. coli strains in 2 mL of LB medium.
The chemically competent strains chosen are as follows:

BL21
DH5α
TOP10

**Protocol for pre-culture preparation**

In a 15 mL tube, add 2 mL of LB medium and inoculate cells from glycerol

## Thursday, 20th June

We started to make 200 mL of BL21 and DH5α E. coli strains and 400 mL of TOP10 to make competent cells.

**Protocol for competent cells**

First prepare the following solutions required to make competent cells:
Solution for 1M Cacl2:
Add 14,30g of CaCl2 into 100 ml desalted water

Solution for 0,1M Cacl2:
Add 50 mL of CaCl2 1M solution into 450 ml of desalted water

Solution for 0,1M Cacl2 + 15% glycerol:
Add 50 mL of CaCl2 1M solution and 75 mL of glycerol 100% into 450 ml of desalted water

For 200 ml LB medium, add 400 µL of strain sample.
Let the bacteria grow until it reaches an OD between 0,3 and 0,35.
Once it reached the right OD, put the medium on ice for 30 minutes to slow down growth.
Split the 200 mL into 4x50 mL tubes then centrifuge at 3000 rpm for 5 minutes at 4°C and suppress supernatant afterwards.
Resuspend the cells with 5 mL of Cacl2 at 0,1M for each 50 mL tube.
Again, put the medium on ice for 30 minutes.
Centrifuge at 3000 rpm for 5 minutes at 4°C then suppress supernatant.
Resuspend the cells with 1 mL of Cacl2 at 0,1M + 15% glycerol for each 50 mL tube.
Split the total 4 mL into 40 tubes containing each 100 µL of concentrated cell solution. This step should be executed fast enough and on ice.

## Friday, 21st June

To check the quality of our work, each strain has been plated on LB medium with ampicillin, kanamycin or chloramphenicol in order to evaluate if it contaminated or not. Furthermore, to evaluate wether our strains are competent or not, we also transformed our bacteria with a pSB1A3 plasmid (red colonies) and plated them on LB medium with ampicillin only.

**Protocol for transformation**

For 100 µL of chemical competent cells, add 1 µL of plasmidic DNA.
Let 30 minutes on ice.
Let the cells at 42°C for exactly 50 seconds.
Let 5 minutes on ice.
Resuspend the cells with 1 mL of LB spread on a petri dish then let it at 37°C.

# Week 2: 24th June - 30th June

## Monday, June 24th

Our competents cells made on friday the 21st were plated and incubated the whole week-end at 30°C. Today, we analyzed the plates and came to the conclusion that the medium was contaminated due to poor protocol execution. As a consequence, we decided to make new LB medium and recalculate the antibiotic concentrations.

**Protocol for LB medium preparation**

Add 20 g of LB broth into 1000 mL of desalted water and autoclave.

**Protocol for antibiotic solutions preparation**

Kanamycin is used as an effective concentration of 10-50 µg/ml and at a storage concentration of 10 mg/ml
Carbenicillin is used as an effective concentration of 20-60 µg/ml and at a storage concentration of 50 mg/ml
Chloramphenicol is used as an effective concentration of 25-170 µg/ml and at a storage concentration of 34 mg/ml (ethanol)
Tetracyclin is used as an effective concentration of 10-50 and at a storage concentration of 5 mg/ml (ethanol)

## Tuesday, June 25th

New competent cells have been made with the same bacterial strains (BL21, DH5α and TOP10). We thought that the contamination of our cells was due to the CaCl2 solution that may not have been sterile and/or incorrect manipulated. As a consequence, we prepared new CaCl2 solutions made sure to autoclave them before usage. We ran the same tests as monday the 24th to evaluate the quality of our work and check for potential contamination.

## Wednesday, June 26th

The results of the competent cells protocol is as follows:
DH5α strain was not contaminated but competent
TOP10 strain was contaminated only on the Kanamycin plate but competent
BL21 strain was completely contaminated and, thus, not usable.

## Thurdsay, June 27th

We finished designing are first two plasmids and constructed the primers to extract the natural promoters from the genomic DNA from E. coli and the different parts for Golden Gate assembly.

## Friday, June 28th

We want to obtain reasonnable competent cells for our oncoming transformation for newt week. Thus, we decided to plate the three strains on LB-Agar medium in order to isolate one and only one colony for monday when we'll start over the whole competent process.

# Week 3: 1st July - 7th July

## Tuesday, July 2nd

We prepared the BL21 and BG1655 strains to be competent.

## Wednesday, July 3rd

The two strains have been contaminated and are not usable.

## Thursday, July 4th

Genomic DNA extraction of the BL21 and BG1655 strains have been done using two different methods.
We received the primers we have ordered. They have been prepared to do a PCR. In order to get our genes of interest we did a PCR using as the DNA template the genomic DNA extracted from BL21 and BG1655.

## Friday, July 5th

To ensure the amplification of our genes by PCR, we did a agarose gel electrophoresis. To do so, we first prepared a TAE 50X stock solution. By diluting it, we use a 1X TAE solution to do a 1% agarose gel. We obtain the following gel. That showed that only two of our genes have been amplificated by PCR. A new PCR will be made.

# Week 4: 8th July - 14th July

## Monday, July 8th

We prepared the BL21 and BG1655 strains to be competent.

## Tuesday, July 9th

|  |  |
| --- | --- |
| ![Petri box](http://2013.igem.org/wiki/images/e/e1/WP_20130711_004.jpg) ![Petri box](http://2013.igem.org/wiki/images/b/b7/WP_20130711_006.jpg) Both strain are not contaminated. | ![Petri box](http://2013.igem.org/wiki/images/f/f1/WP_20130711_002.jpg) ![Petri box](http://2013.igem.org/wiki/images/c/cf/WP_20130711_003.jpg) BL21 are highly competent, BG1655 are little competent. |

## Wednesday, July 10th

### PCR procedure optimization

**Aim: Define the number of matrix of optimal DNA to amplify a gene by PCR.**

**Data:**

* [primer] = --- ng/µl
  * size(primer) = --- nt

For PCR we chose to use … ng of primer (V=…µL). As we know the size of our primers, we could define the number of primers that we have to use to begin the reaction.
We define the number of recquired matrix DNA in terms of concentration of sample with the following formula:

Analysis of Agarose gel's results:

### Extraction of natural promoter sequence

**Aim:** We want to have sequences genes' promoters' sequences under the control of FUR (Ferric Uptake Regulation)transcription factor.

We remake PCR samples that failed the previous week.

* Fec A* Ent C* Fec C* Ace B

PCR products are placed on gel and purificate. After, PCR products are tested with nanodrop.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **Fec A** (BG1655)  [c] = 23.7 ng/µl  260/280 = 1.90 | **Fec A** (BL21)  [c] = 53.1 ng/µl  260/280 = 1.73 | **Ent C** (BG1655)  [c] = 52.4 ng/µl  260/280 = 1.71 | **Ent C** (BL21)  [c] = 50.0 ng/µl  260/280 = 1.94 | **Ace B** (BG1655)  [c] = 46.5 ng/µl  260/280 = 1.89 | **Fec C** (BG1655)  [c] = 34.2 ng/µl  260/280 = 1.91 |

![pcr 10-07](http://2013.igem.org/wiki/images/9/9b/PCR_10-07.png)