iGEM 2013 — Team Paris_Bettencourt

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# Team:Paris Bettencourt/Notebook

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* [PROJECT](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Overview "Project Overview")

  + [OVERVIEW](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Overview "Project Overview")
  + [DETECT](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect "Detect")
  + [TARGET](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Target "Target")
  + [INFILTRATE](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Infiltrate "Infiltrate")
  + [SABOTAGE](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Sabotage "Sabotage")
* [ACHIEVEMENTS](https://2013.igem.org/Team%3AParis_Bettencourt/Results "Achievements")

  + [PARTS](https://2013.igem.org/Team%3AParis_Bettencourt/Parts "Submitted Parts")+ [RESULTS](https://2013.igem.org/Team%3AParis_Bettencourt/Results "Results")
    + [MEDAL FULFILLMENT](https://2013.igem.org/Team%3AParis_Bettencourt/Medal_Fulfillment "Medal Fulfillment")
* [HUMAN PRACTICE](https://2013.igem.org/Team%3AParis_Bettencourt/Human_Practice/Overview "Human Practice Overview")

  + [OVERVIEW](https://2013.igem.org/Team%3AParis_Bettencourt/Human_Practice/Overview "Human Practice Overview")
  + [GENDER STUDY](https://2013.igem.org/Team%3AParis_Bettencourt/Human_Practice/Gender_Study "Gender Study")
  + [TECHNOLOGY TRANSFER](https://2013.igem.org/Team%3AParis_Bettencourt/Human_Practice/Technology_Transfer "Technology_Transfer")
  + [TB FACTS](https://2013.igem.org/Team%3AParis_Bettencourt/Human_Practice/TB_Facts "TB Tacts")
  + [TB IN FRANCE](https://2013.igem.org/Team%3AParis_Bettencourt/Human_Practice/TB_France "TB in France")
  + [TB GALLERY](https://2013.igem.org/Team%3AParis_Bettencourt/Human_Practice/TB_Gallery "TB Gallery")
* [SAFETY](https://2013.igem.org/Team%3AParis_Bettencourt/Human_Practice/Safety "Safety")
* [COLLABORATION](https://2013.igem.org/Team%3AParis_Bettencourt/Collaboration "Collaboration")

  + [COLLABORATION](https://2013.igem.org/Team%3AParis_Bettencourt/Collaboration "Collaboration")
  + [SENSIGEM](https://2013.igem.org/Team%3AParis_Bettencourt/SensiGEM "SensiGEM")
* [NOTEBOOK](https://2013.igem.org/Team%3AParis_Bettencourt/Notes "Notebook")

  + [DAY NOTES](https://2013.igem.org/Team%3AParis_Bettencourt/Notebook "Day Notes")
  + [PROTOCOLS](https://2013.igem.org/Team%3AParis_Bettencourt/Protocols "Protocols")
  + [BIBLIOGRAPHY](https://2013.igem.org/Team%3AParis_Bettencourt/Bibliography "Bibliography")
* [TEAM](https://2013.igem.org/Team%3AParis_Bettencourt/Team "Team")

  + [OUR TEAM](https://2013.igem.org/Team%3AParis_Bettencourt/Team "Team")
  + [GALLERY](https://2013.igem.org/Team%3AParis_Bettencourt/Gallery "Team Gallery")
  + [ATTRIBUTIONS](https://2013.igem.org/Team%3AParis_Bettencourt/Attributions "Attributions")
  + [ACKNOWLEDGEMENTS](https://2013.igem.org/Team%3AParis_Bettencourt/Acknowledgements "Acknowledgements")

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## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Monday 22nd July

***Culture purification by streak plate method and preparing the glycerol stock for the sSP001 (TN 57) and sSP002 (NEC) strains.***

Place your note here

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Tuesday 23rd July

***Colony PCR***

· Strain: Keio ΔpyrF
· Primers: JW182 and JW183
· Amplified region: kanamycine resistance gene

![](https://static.igem.org/mediawiki/2013/b/b3/13%EF%80%A207%EF%80%A224_first_run_KanR.jpg)

![](https://static.igem.org/mediawiki/2013/9/91/13%EF%80%A207%EF%80%A224_second_run_KanR.jpg)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Monday 29th July

Liquid culture of:
pCOLA DUET (from glycerol stock of Sabotage Group) - 20 ml culture
pSSB1 (from plate) - 5 ml culture
pSSB2 (from plate ) - 5 ml culture

Plating of addgene bacteria containing the plasmids we want:
streak out bacteria from addgene to get single colonies:
pBAC-BA-lacZ
pCRISPR
pCas9
pCRIPSR::rpsL

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Tuesday 30th July

***Liquid culture, Miniprep and Glycerol stock***

**Liquid culture of:**
pCOLA DUET (from liquid culture (29.07.13)) - 20 ml culture
pSSB1 (from liquid culture (29.07.13)) - 5ml culture
pSSB2 (from liquid culture (29.07.13)) - 5 ml culture
pBAC-BA-lacZ (from plate ) - 5 ml culture
pCRISPR (from plate ) - 5 ml culture
pCas9 (from plate ) - 5 ml culture
pCRIPSR::rpsL (from plate ) - 5 ml culture
F+ strain - (from glycerol stock Sabotage) - 10 ml culture
Keio Keio delta pyrF (from plate ) - 10 ml culture

**Miniprep (pCOLA-DUET = sSP007) using the Thermo Scientific Miniprep Kit**

1) Pellet 2x9 ml of liquid culture (4000 rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250 µL of resuspension solution
4) add 250 µL of lysis solution, mix by inverting 4-6 times
5) add 350 µL of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 µL wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove left over liquid
10) transfer the column on a 1.5 ml tube
11) add 50 µL of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

pSP001= pCOLA-DUET: 65 ng/ul

**Glycerol Stock**
from overnight culture of MG1655-pCOLA-DUET (sSP007)
Centrifuge 4000 rpm, 10 minutes,
take out liquid
resuspend cells in 0,5 mL glycerol (60%) , 2 mL LB
freeze in -80°C

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Wednesday 31st July

***Conjugation, Miniprep addgene plasmids,Glycerol Stock of addgene plasmids***

**Conjugation**

sSP001, SP002, keio Delta PyrF
F+ comes from XL1 Blue (glycerol stock Sabotage) => F+ is tetR

Protocol :
1) From O/N cultures Dilute strains 1/100 in LB
2) Wait for OD to reach O,2
3) Prepare 4 tubes (in BD tubes) :
- Tube 1 = 0,5 mL LB with Strain 1 (sSP001) ,5 mL LB = control
- Tube 2 = 0,5 mL LB with Strain 2 (sP001) + 0,5 mL LB = control
- Tube 3 = 0,5 mL LB with Strain 3 (keio Delta pyrF) + 0,5 mL LB = control
- Tube 4 = 0,5 mL LB with Strain 4 (XL1) + 0,5 mL LB = control
- Tube 5 = 0,5 mL LB with Strain 1 (sSP001) + 0,5 mL LB with Strain 4 (XL1)
- Tube 6 = 0,5 mL LB with Strain 2 (sSP002) + 0,5 mL LB with Strain 4 (XL1)
- Tube 7 = 0,5 mL LB with Strain 3 (keio Delta pyrF) + 0,5 mL LB with Strain 4 (XL1)
4) Incubate 2 hours at 37°C (actually not in the shaker, but we accidently kept them in the shaker...)
5) Plate 10ul for controls, 100uL, 10ul for mixed tubes on LB antibiotics (Tube 5: Chl + Tet, Tube 6: Chl + Tet, Tube 7: Kan + Tet)
6) Incubate overnight at 37°C

**Miniprep addgene plasmids**
1) Pellet 4 ml of liquid culture (4000 rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250 µL of resuspension solution
4) add 250 µL of lysis solution, mix by inverting 4-6 times
5) add 350 µL of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 µL wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove leftover liquid
10) transfer the column on a 1.5 ml tube
11) add 50 µL of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

See Database for plasmid reference
pCas9:
pCRISPR:
pBac-LacZ:
pCRISPR::rpsL:

**Glycerol Stock of addgene plasmids**
 (see Database for strain reference)
from overnight culture
Centrifuge 4000 rpm, 10 minutes,
take out liquid
resuspend cells in 0,5 mL glycerol (60%) , 2 mL LB
freeze in -80°C

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Thursday 1st August

***PCR (gradient + usual) of SPCR1, SPCR7, SPCR8, SPCR10, SPCR9, SPCR4***

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
|  | **1x** |
| Nuclease-free water | 37.25 µL |
| 5x Phusion HF Buffer | 10 µL |
| 10 mM dNTPs | 1 µL |
| Forward Primer (10 µM) | 0.5 µL |
| Reverse Primer (10 µM) | 0.5 µL |
| Template Plasmid | 0.25 µL |
| Phusion DNA Polymerase | 0.5 µL |
| **Total Volume** | 50 µL |

Gradient:
Pos 1: 40°
Pos 2: 40,2°
Pos 3: 41,3°
Pos 4: 43,1°
Pos 5: 45,4°
Pos 6: 48°
Pos 7: 50,7°
Pos 8: 53,5°
Pos 9: 56,0°
Pos 10: 58,1°
Pos 11: 59,8°C
(Pos 12: 60,6°)

Usual PCR: 52°

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Friday 2nd August

***Place your twit here***

Gels with PCR products: 1% Agar
100V 20 Min
10 min staining in BET

![](https://static.igem.org/mediawiki/2013/d/d7/13%EF%80%A208%EF%80%A202_SCPR1_iS008_iS009.jpg)

![](https://static.igem.org/mediawiki/2013/3/3e/13%EF%80%A208%EF%80%A202_SPCR4_iS0018_iS019.jpg)

![](https://static.igem.org/mediawiki/2013/f/f4/13%EF%80%A208%EF%80%A202_SPCR7_iS010_iS011.jpg)

![](https://static.igem.org/mediawiki/2013/0/01/13%EF%80%A208%EF%80%A202_SPCR8_iS012_iS013_2.jpg)

![](https://static.igem.org/mediawiki/2013/f/f9/13%EF%80%A208%EF%80%A202_SPCR9_iS016_iS017.jpg)

![](https://static.igem.org/mediawiki/2013/f/fd/13%EF%80%A208%EF%80%A202_SPCR10_iS014_iS015.jpg)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Monday 5th August

***Place your twit here***

**5ml liquid culture with proper antibiotics of:**
SSp001 + F+
sSP002 + F+
keio delta pyrF + F+

**Gelelectrophoresis:**
1% Agar, 100 V 20 min

![](https://static.igem.org/mediawiki/2013/c/cf/13%EF%80%A208%EF%80%A205_SPCR7_1%2C2%2C10_SPCR4_1%2C2%2C4%2C10%2C11_SCPR10_1%2C4%2C5%2C6%2C11.jpg)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Tuesday 6th August

***Liquid cultures of sSP008-sSP0011***

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Wednesday 7th August

***Glycerol Stock of sSP008-sSP0011, M13 Test, Single Colonies, Liquid cultures, PCR and Transformation***

**Glycerol Stock of sSP008-sSP0011**
Take 1.5ml of ON culture + 500ul Glycerol (60%)
Freeze at -80°C

**M13 Test**
Plate from ON culture:
1) 1:1000 - sSP012: sSP009 (plate 200ul)
2) 200 ul sSP012
3) 200ul sSp009

**Single Colonies**
streak out ON culture of sSP012 to get single colonies

**Liquid cultures** of pir+ E.coli and pKD13 from Jake

**PCR of SPCR4, SPCR7, SPCR8, SPCR9, SPCR10 from circuar backbone, directly taken from Biobrick plate**

Protocol

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
|  | **1x** |
| Nuclease-free water | 37,25µL |
| 5x Phusion HF Buffer | 10µL |
| 10 mM dNTPs | 1µL |
| Forward Primer (10 µM) | 0.5µL |
| Reverse Primer (10 µM) | 0.5µL |
| Template Plasmid | 0.25µL |
| Phusion DNA Polymerase | 0.5µL |
| **Total Volume** | 50µL |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Thermocycler Protocol: NEB Phusion** |  |  |  |  |
|  | Temp | Time |  |  |
| Start | 98°C | 30 sec | Melt |  |
|  |  |  |  |  |
| Cycle 1 | 98°C | 5 sec | Melt | 35 cycles |
| Cycle 2 | 40.5°C / 60°C | 25 sec | Anneal |  |
| Cycle 3 | 72°C | 30 sec per kb | Extend |  |
|  |  |  |  |  |
| Finish | 72°C | 5 min | Extend |  |
| Store | 10°C | Forever | Store |  |

**Transformation** Biobricks BBa\_K649304 (with pSB1C3 backbone) and BBa\_J04450 (with pSB1A3 backbone) into NEB Turbo (heat shock trafo)

1) Thaw competent cells on ice.
2) Place 20 ul of cells in a pre-chilled Eppendorf tube.
3) Add 2.5 ul of plasmid (from Biobrick stock)
4) Mix gently by flicking the tube.
5) Chill on ice for 10 minutes.
4) Heat shock at 42 °C for 30 seconds
5) Return to ice for 2 minutes.
6) Add 200 ul LB medium and recover the cells by shaking at 37 °C.
Ampicillin: 15-30 minutes
Chloramphenicol: 60-120 minutes
7) Plate out the cells on selective LB (10ul)
8) Incubate at 37 °C. Transformants should appear within 12 hrs.

**Result**: BBa\_J04450: many colonies, BBa\_K649304 no colonies, plate rest

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Thursday 8th August

***Liquid culture of FR-433 (M13)***

Gel of PCR products. 100V 20min 1%

4-7-8-9-10 (40.5°C)- 4-7-8-9-10 (60°C)
![](https://static.igem.org/mediawiki/2013/f/fc/13%EF%80%A208%EF%80%A208_SPCR4_SPR7_SPCR8_SCPR9_SPCR10.jpg)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Friday 9th August

***Liquid culture, Gel,
Trafo of BBa\_E1010 (pSB1C3 backbone) into NEB turbo, Glycerol Stock, Making electro competent cells of pir+ E.coli, Transforming pKD13 into pir+ E.coli, Miniprep NEB turbo with BBa-J04450:, Liquid culture and Patch plates.***

**Liquid culture**
1:1000 from liquid: FR-433 (M13), 10 ml
pir+ E.coli: from plate, 25 ml
XL10: from plate 10 ml

**Gel**: rest of circular backbone PCR product, 1% Agar, 100V, 20 min
SPCR4-7-8-9-10 (40.5) SPCR4-7-8-9-10 (60.5)
Gel picture:
didn’t work

**Trafo of BBa\_E1010 (pSB1C3 backbone) into NEB turbo**
1) Thaw competent cells on ice.
2) Place 20 µl of cells in a pre-chilled Eppendorf tube.
3) Add 2.5 µl of plasmid (from Biobrick stock)
4) Mix gently by flicking the tube.
5) Chill on ice for 10 minutes.
4) Heat shock at 42 °C for 30 seconds
5) Return to ice for 2 minutes.
6) Add 200 µl LB medium and recover the cells by shaking at 37 °C.
Chloramphenicol: 60-120 minutes
7) Plate out the cells on selective LB (10ul)
8) Incubate at 37 °C. Transformants should appear within 12 hrs.

**Glycerol Stock FR-433 (M13) -> sSP012**
Glycerol Stock pir+ -> sSP013
Glycerol Stock pKD13 -> sSP014
Glycerol Stock NEB turbo with BBa-J04450 -> sSP015
Take 1.5 ml of ON culture + 500ul Glycerol (60%)
Freeze at -80°C

**Making electro competent cells of pir+ E.coli**
Preparation of Electrocompetent Cells
Note: Competent cells should never be vortexed, as this will cause them to lyse and release salts into the media. Resuspend cells by pipetting up and down with a large pasteur pipet. Once they are chilled, cells should be continuously cold.

1) The night before the transformation, start an overnight culture of cells.
pir+ E.coli
2) The day of the transformation, dilute the cells 100X.
100 ml LB
Grow at 30°C for about 90 minutes.
3) When the cells reach an OD600 of 0.2.
4) Harvest the cells.
When the cells reach an OD600 of between 0.6 and 0.8.
Split the culture into 2x 50 ml falcon tubes, on ice.
Centrifuge at 4 °C for 10 min at 4000 rpm.
5) Wash and combine the cells.
Remove the supernatant.
Resuspend the cells in 2x 25 ml of ice cold water.
Combine the volumes in a single 50 ml falcon tube.
6) Wash the cells 2 more times.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 50 ml of ice cold water.
Repeat.
7) Wash and concentrate the cells for electroporation.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 1-2 ml of ice cold water.
We will use 200 µl of washed cells per transformation.

**Transforming pKD13 into pir+ E.coli**
1) Prepare BD tubes with a pipette filled with LB at the interior of each tube (pipette supplied with the electroporation cuvette)

2) Test the purity of the electrocompetent cells.
Add 200 ul of washed cells to a cuvette.

3) Mix the cells and DNA in a cuvette.
200 ul of washed cells with 200 ng of PCR product.
Keep the cuvette on ice until just before the electroporation.

4) Preload a pipette with 1 ml of LB.

5) Pulse the cuvette with voltage.
Dry the electrodes with a kimwipe prior to loading.
Use the EC2 setting.

6) Listen for arcing.
A cracking sound means all the cells are dead.
Note the time constant: 5 is good, 5.8 is great.

7) Immediately recover the cells.
Add the 1 ml of preloaded LB and pipet up and down to mix.
Collect 1 ml of cells, some volume is lost in the cuvette.

8) Incubate 2 h at 37 °C with shaking.

9) Plate 100 ul of recovered cells on selective plates.
Use antibiotic appropriate to the part being integrated.
Let the other 900 ul rest overnight at room temperature.

10) Concentrate and plate the remaining cells
Spin down quickly and resuspend in 100 ul LB before plating.

Transformed cells should be incubated at 37 °C.
Colonies should appear 24-48 h after plating.

**Miniprep NEB turbo with BBa-J04450:**
1) Pellet 2x 9 ml of liquid culture (4000rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250 µl of resuspension solution
4) add 250 µl of lysis solution, mix by inverting 4-6 times
5) add 350 µl of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 µl wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove left over liquid
10) transfer the column on a 1.5 ml tube
11) add 50ul of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

J04450: 0ng/ul

**Liquid culture of:**
sSP012
sSP008
sSP009
sSP010
sSP011
sSP001
sSP002
XL-10 = sSP016
sSP017= Litmus

**Patch plates of:**
sSP010
sSP013
sSP009s
sSP014
sSP012

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Saturday 10th August

***Test of M13 and Liquid culture***

**Test of M13**

1) 100 μ l of plating bacteria per tube
2) Prepare tenfold serial dilutions (10-6 to 10-9 ) of the bacteriophage stock in LB
3) Put 10 μ l/ 100 μ l of each dilution to the bacteria
4) Mix the bacteriophage particles with the bacterial culture by vortexing gently.
5) Add 40 μ l of X-gal solution (working conc) and IPTG (working conc) solution to each of the tubes
6) Add 3ml of Agar to each tube and gently vortexing for 3 seconds
7) Pour the mixture onto plates containing LB medium supplemented with 5 mM MgCl2
8) Swirl the plate gently to ensure an even distribution of bacteria and agar.
9) Incubate them at 37°C.

**Liquid culture:**
E1010
J04550

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Sunday 11th August

***Patches of sSP001-sSP008, sSP015-18 and Stress experiment (Plate reader)***

**Patches of sSP001-sSP008, sSP015-18**

**Stress experiment (Plate reader)**
sSP001, sSP002, sSP010, sSP011, sSP012 (M13)
1:1000 bacteria in M9 Glucose
Mitomycin C concentrations: 3,3\*10^-3, 1,6\*10°-3, 6,6\*10°-4, 3,3\*10°-4
Quadruplets of bacteria (+phage), Triplets of MMC

See also pipetting scheme in folder Experiments on Dropbox

**On culture of FR-433, XL-10, delta pyrF**
1:1000 culture at 30°

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Monday 12th August

***Glycerol Stock, Miniprep, M13 plate test, Patches***

**Glycerol Stock**
sSP018 - NEB with E1010
sSP017
sSP016
Take 1.5ml of ON culture + 500ul Glycerol (60%)
Freeze at -80°C

**Miniprep**
E1010
J04450

1) Pellet 2x 2ml of liquid culture (4000rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250ul of resuspension olution
4) add 250ul of lysis solution, mix by inverting 4-6 times
5) add 350ul of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 ul wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove left over liquid
10) transfer the column on a 1.5ml tube
11) add 50ul of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

J04450: ng/ul
E1010: ng/ul

**M13 plate test**
1) 100 μ l of plating bacteria per tube
2) Prepare tenfold serial dilutions (10-6 to 10-9 ) of the bacteriophage stock in LB
3) Put 10 μ l/ 100 μ l of each dilution to the bacteria
4) Mix the bacteriophage particles with the bacterial culture by vortexing gently.
5) Add 40 μ l of X-gal solution (working conc) and IPTG (working conc) solution to each of the tubes
6) Add 3ml of Agar to each tube and gently vortexing for 3 seconds
7) Pour the mixture onto plates containing LB medium supplemented with 5 mM MgCl2
8) Swirl the plate gently to ensure an even distribution of bacteria and agar.
9) Incubate them at 37°C.

**Patches**
check them and prepare new ones of those that haven’t been done yet

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Tuesday 13th August

***PCR circular and linear 40.5°C/ 60°C, Conjugation of XL-10 (with sSP011), Patches, Digestion M13 backbone, RFP insert and Gel of PCR***

**PCR circular and linear, 40.5°C, 60°**
of pSB1A3, pSB1C3

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
|  | **1x** |
| Nuclease-free water | 37,25µL |
| 5x Phusion HF Buffer | 10µL |
| 10 mM dNTPs | 1µL |
| Forward Primer (10 µM) | 0.5µL |
| Reverse Primer (10 µM) | 0.5µL |
| Template Plasmid | 0.25µL |
| Phusion DNA Polymerase | 0.5µL |
| **Total Volume** | 50µL |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Thermocycler Protocol: NEB Phusion** |  |  |  |  |
|  | Temp | Time |  |  |
| Start | 98°C | 30 sec | Melt |  |
|  |  |  |  |  |
| Cycle 1 | 98°C | 5 sec | Melt | 35 cycles |
| Cycle 2 | 40.5°C / 60°C | 25 sec | Anneal |  |
| Cycle 3 | 72°C | 30 sec per kb | Extend |  |
|  |  |  |  |  |
| Finish | 72°C | 5 min | Extend |  |
| Store | 10°C | Forever | Store |  |

**Conjugation of XL-10 (with sSP011)**
1) From O/N cultures Dilute strains 1/100 in LB
2) Wait for OD to reach O,2
3) Prepare tube (in BD tubes) :
- Tube = 0,5mL LB with Strain (sSP011) + 0,5mL LB with Strain (XL-10 Kan)
4) Incubate 2 hours at 37°C (actually not in the shaker, but we accidently kept them in the shaker...)
5) Plate 20ul for mixed tube on LB antiobiotics (Tet, Kan)
6) Incubate overnight at 37°C

**Patches**
check and make new ones

**Digestion M13 backbone, RFP insert**
for Backbone (M13mp18 plasmid): 3ug
7,58 ul plasmid (c=395ng/ul)
3 ul EcoRI
3 ul PstI
3ul 10x Fast Digest
13,42 ul H20
incubate for 12 min on 37°
heat inactivation: 80° 5 min
for insert (BBa\_J04450): 5ug
31,4 ul plasmid
5 ul EcoRi
5 ul PstI
3,6 ul H20
incubate for 20 min at 37°
heat inactivation: 80° 5min

**Gel of PCR (Amp 40.5 circ, lin, Chl 60° lin, circ)**
100V, 20 min 1% gel

![](https://static.igem.org/mediawiki/2013/5/5d/13%EF%80%A208%EF%80%A213_SPCR_AmpR_backbones_40.5%C2%B0_60%C2%B0_circular_linear_4%2C7%2C8%2C9%2C10.jpg)

![](https://static.igem.org/mediawiki/2013/2/27/13%EF%80%A208%EF%80%A213_SPCRs_ChlR_backbones_40.5%C2%B0_60%C2%B0_circular_linear_4%2C7%2C8%2C9%2C10.jpg)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Wednesday 14th August

***Gel of PCR, Gel of Digest, Gel Extraction, DpnI digest of PCR products, PCR purification***

**Gel of PCR** (Amp 60° circ, lin, Chl 40.5 circ, lin)
100V, 20 min 1% gel

**Gel of Digest**
100V, 20 min 1% gel

![](https://static.igem.org/mediawiki/2013/1/1a/13%EF%80%A208%EF%80%A214_SPCR_AmpR_backbones_40.5%C2%B0_60%C2%B0_circular_linear_4%2C7%2C8%2C9%2C10.jpg)

**Gel Extraction**
Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 μl). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
Add 1 gel volume of isopropanol to the sample and mix (actually only needed for very small products or very big products)
To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
Discard flow-through and place QIAquick column back in the same collection tube.
To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).
12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.

**DpnI digest of PCR products:**
SPCR4 (40ul) - add 1 ul DpnI
SPCR 7 (150ul) - add 3 ul of DpnI
SPCR8 (60 ul) - add 3 ul of DpnI
SPCR 9 (150ul) - add 3 ul of DpnI
SPCR10 (110ul) - add 3 ul of DpnI
Incubate for 15 min at 37°C

**PCR purification**
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Thursday 15th August

***Liquid cultures***

**Liquid cultures**:
sSp009
sSP012
sSP016
sSp020
Xgal Bacteria from Aude

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Friday 16th August

***Liquid M13 Test, Glycerol Stock, Miniprep***

**Liquid M13 Test**

OD (sSP020=XL10 KanR with F+)=0,557
OD (sSP016=XL10 KanR)=0,515
OD (sSP009= delta pyrF with F+)=0,561
OD(sSP012=FR-433=M13)=0,695

Prepare Falcons containing 500ul of each strain, adding 500ul of 10^-6 dilution of FR-433
add 1ul of IPTG and 1ul of Xgal
Incubate at 37° on shaker

**Glycerol Stock**
sSP019 (pir+ E.coli transformed with pKD13)
sSP020 (XL10 KanR with F+)

**Miniprep**
sSP019
sSP017

1) Pellet 2x 2ml of liquid culture (4000rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250ul of resuspension olution
4) add 250ul of lysis solution, mix by inverting 4-6 times
5) add 350ul of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 ul wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove left over liquid
10) transfer the column on a 1.5ml tube
11) add 50ul of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

pKD13: 311ng/ul
Litmus 28i: 93ng/ul

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Monday 19th August

***Restreak XL-1 Blue Litmus28i, PCR, PCR purification Backbone (M13mp18), Ligation (RFP into M13mp18 backbone), Trafo of Ligation product (RFP in M13mp18 plasmid) into NEB turbo and Test Xgal***

**Restreak XL-1 Blue Litmus28i**

**PCR**

SPCR5, SPCR6, 49,3°C

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
|  | **1x** |
| Nuclease-free water | 37,25µL |
| 5x Phusion HF Buffer | 10µL |
| 10 mM dNTPs | 1µL |
| Forward Primer (10 µM) | 0.5µL |
| Reverse Primer (10 µM) | 0.5µL |
| Template Plasmid | 0.25µL |
| Phusion DNA Polymerase | 0.5µL |
| **Total Volume** | 50µL |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Thermocycler Protocol: NEB Phusion** |  |  |  |  |
|  | Temp | Time |  |  |
| Start | 98°C | 30 sec | Melt |  |
|  |  |  |  |  |
| Cycle 1 | 98°C | 5 sec | Melt | 35 cycles |
| Cycle 2 | 40.5°C / 60°C | 25 sec | Anneal |  |
| Cycle 3 | 72°C | 30 sec per kb | Extend |  |
|  |  |  |  |  |
| Finish | 72°C | 5 min | Extend |  |
| Store | 10°C | Forever | Store |  |

**PCR purification Backbone (M13mp18)**
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.

**Ligation (RFP into M13mp18 backbone)**
3ul Backbone
14 ul Insert
2ul 10x T4 DNA Ligase HC Buffer
1 ul T4 DNA Ligase

Incubate for at least 10min at 22°C

**Trafo of Ligation product (RFP in M13mp18 plasmid) into NEB turbo**
1) Thaw competent cells on ice.
2) Place 20 ul of cells in a pre-chilled Eppendorf tube.
3) Add 3 ul of plasmid (from Biobrick stock)
4) Mix gently by flicking the tube.
5) Chill on ice for 10 minutes.
4) Heat shock at 42 °C for 30 seconds
5) Return to ice for 2 minutes.
6) Add 200 ul LB medium and recover the cells by shaking at 37 °C.
7) Plate out the cells on LB (200ul)
8. Incubate at 37 °C. Transformants should appear within 12 hrs.

**Test Xgal**
3ml of Liquid culture with
1) Xgal from iGEM 2012 (Jake) (+IPTG)
2) Xgal from last week (in DMF) (+IPTG)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Tuesday 20th August

***Gel of PCR and Xgal Test***

**Gel of PCR**

1%, 100V, 20min

![](https://static.igem.org/mediawiki/2013/a/a2/13%EF%80%A208%EF%80%A220_at_49.3C_SPCR5_iS005_iS007_SPCR6_iS005_iS006.jpg)

Result: PCR didn’t work

**Xgal Test**

MG1655 (of Jake) in liquid culture with Xgal of iGEM 2012, Aude and new one (Anne)

Result:
All cultures turn blue (Aude, less blue)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Wednesday 21st August

***Gradient PCR SPCR5, SPCR6, M13 Test, Electrocompetent Cells (sSP017-XL-1 Blue, Litmus 28i), Transformation***

**Gradient PCR SPCR5, SPCR6**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
|  | **1x** |
| Nuclease-free water | 37,25µL |
| 5x Phusion HF Buffer | 10µL |
| 10 mM dNTPs | 1µL |
| Forward Primer (10 µM) | 0.5µL |
| Reverse Primer (10 µM) | 0.5µL |
| Template Plasmid | 0.25µL |
| Phusion DNA Polymerase | 0.5µL |
| **Total Volume** | 50µL |

Gradient:
Pos 1: 40.1°
Pos 2: 40,8°
Pos 3: 42.2°
Pos 4: 44.2°
Pos 5: 46.7°
Pos 6: 49.4°
Pos 7: 52.1°
Pos 8: 54.7°
Pos 9: 57.1°
Pos 10: 59.0°
Pos 11: 60.2°C

**M13 Test**
Liquid cultures of sSP012 and sSP020
1) 100 μ l of plating bacteria per tube
2) Prepare tenfold serial dilutions (10-6 to 10-9 ) of the bacteriophage stock in LB
3) Put 10 μ l/ 100 μ l of each dilution to the bacteria
4) Mix the bacteriophage particles with the bacterial culture by vortexing gently.
5) Add 40 μ l of X-gal solution (working conc) and IPTG (working conc) solution to each of the tubes
6) Add 3ml of Agar to each tube and gently vortexing for 3 seconds
7) Pour the mixture onto plates containing LB medium supplemented with 5 mM MgCl2
8) Swirl the plate gently to ensure an even distribution of bacteria and agar.
9) Incubate them at 37°C.

**Electrocompetent Cells (sSP017-XL-1 Blue, Litmus 28i)**
1) The night before the transformation, start an overnight culture of cells.
sSP017 (XL1 Blue Litmus 28i)
2) The day of the transformation, dilute the cells 100X.
100 ml LB Amp.
Grow at 30°C for about 90 minutes.
3) When the cells reach an OD600 of 0.2.
4) Harvest the cells.
When the cells reach an OD600 of between 0.6 and 0.8 (OD: 0,683)
Split the culture into 2x 50 ml falcon tubes, on ice.
Centrifuge at 4 °C for 10 min at 4000 rpm.
5) Wash and combine the cells.
Remove the supernatant.
Resuspend the cells in 2x 25 ml of ice cold water.
Combine the volumes in a single 50 ml falcon tube.
6) Wash the cells 2 more times.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 50 ml of ice cold water.
Repeat.
7) Wash and concentrate the cells for electroporation.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 1-2 ml of ice cold water.
We will use 200 ul of washed cells per transformation.

**Transforming pKD13 into pir+ E.coli**
1) Prepare BD tubes with a pipette filled with LB at the interior of each tube (pipette supplied with the electroporation cuvette)

2) Test the purity of the electrocompetent cells.
Add 200 ul of washed cells to a cuvette.

3) Mix the cells and DNA in a cuvette.
200 ul of washed cells with 200 ng of PCR product.
Keep the cuvette on ice until just before the electroporation.

4) Preload a pipette with 1 ml of LB.

5) Pulse the cuvette with voltage.
Dry the electrodes with a kimwipe prior to loading.
Use the EC2 setting.

6) Listen for arcing.
A cracking sound means all the cells are dead.
Note the time constant: 5 is good, 5.8 is great.

7) Immediately recover the cells.
Add the 1 ml of preloaded LB and pipet up and down to mix.
Collect 1 ml of cells, some volume is lost in the cuvette.

8) Incubate 2 h at 37 °C with shaking.

9) Plate 100 ul of recovered cells on selective plates.
Use antibiotic appropriate to the part being integrated.
Let the other 900 ul rest overnight at room temperature.

10) Concentrate and plate the remaining cells
Spin down quickly and resuspend in 100 ul LB before plating.

Transformed cells should be incubated at 37 °C.
Colonies should appear 24-48 h after plating.

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Thursday 22nd August

***Gel of gradient PCR product (Pictures 6,5,5), PCR purification and pooling, Digestion M13 backbone, RFP insert, Test Digestion in Gel, Gel Extraction, Liquid culture, Purification of Backbone Cloning and Miniprep sSP015.***

**Gel of gradient PCR product**
1%, 100V, 20 min

![](https://static.igem.org/mediawiki/2013/5/56/13%EF%80%A208%EF%80%A222_Degest_BBaJ%294550_with_EcoRI_PstI.jpg)

![](https://static.igem.org/mediawiki/2013/e/e8/13%EF%80%A208%EF%80%A222_SPCR_6_iS005_iS006.jpg)

![](https://static.igem.org/mediawiki/2013/d/d3/13%EF%80%A208%EF%80%A222_SPCR5_iS005_iS007.jpg)

**PCR purification and pooling**
Pool all samples that worked
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.

c(SPCR5)= ng/ul
c(SPCR6)= ng/ul

**Digestion M13 backbone, RFP insert**

Backbone/Insert (double the amount)

|  |  |
| --- | --- |
| Regent | Volume |
| Purified Plasmid | 20 ul |
| H2O | 65 ul |
| 10x FastDigest Buffer | 10 ul |
| FastDigest Enzyme 1 (EcoRI) | 2,5 ul |
| FastDigest Enzyme 2 (PstI) | 2,5 ul |
| FastAP Phosphatase | 0 ul |
| **Total Volume** | **100ul** |

Digest for 2.5 hours at 37 °C with shaking.

**Test Digestion in Gel**
1%, 100V, 20 min

**Gel Extraction**
Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 μl). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
Add 1 gel volume of isopropanol to the sample and mix (actually only needed for very small products or very big products)
To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
Discard flow-through and place QIAquick column back in the same collection tube.
To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).
12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.

c=13,6ng/ul

Liquid culture
sSP015

**Purification of Backbone Cloning**
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 30 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.

**Miniprep sSP015**
1) Pellet 2x 2ml of liquid culture (4000rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250ul of resuspension solution
4) add 250ul of lysis solution, mix by inverting 4-6 times
5) add 350ul of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 ul wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove left over liquid
10) transfer the column on a 1.5ml tube
11) add 50ul of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Friday 23rd August

***Stress Experiment (sSP001, sSP002, sSP010, sSP011, M13, MMC), Microscope, Gradient PCR of SPCR2, Digestion of SPCR 5 and SPCR7 and Purification of Digestion.***

**Stress Experiment (sSP001, sSP002, sSP010, sSP011, M13, MMC)**
Dilute ON culture 1:100
Centrifuge (1 min, 10rpm)
Wash 1x with 70% of Volume M9 Glucose
Centrifuge again
Take up in 70% of the VOlume M9 Gluo
100ul in each 96 well plate (see pipetting file)
Add MMC 10ug/ml in corresponding wells and incubate in the plate reader
After 90 min add phages into correscponding wells
put back on the plate reader

**Microscope**
look at the shape and fluorescence at t=0 of sSP001
look at the shape and fluorescence at=90 mi of sSP001 + 10ug/ml MMC
take photos

**Gradient PCR of SPCR2**
iSP001 (gblock) and iS002

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
|  | **1x** |
| Nuclease-free water | 37,25µL |
| 5x Phusion HF Buffer | 10µL |
| 10 mM dNTPs | 1µL |
| Forward Primer (10 µM) | 0.5µL |
| Reverse Primer (10 µM) | 0.5µL |
| Template Plasmid | 0.25µL |
| Phusion DNA Polymerase | 0.5µL |
| **Total Volume** | 50µL |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Thermocycler Protocol: NEB Phusion** |  |  |  |  |
|  | Temp | Time |  |  |
| Start | 98°C | 30 sec | Melt |  |
|  |  |  |  |  |
| Cycle 1 | 98°C | 5 sec | Melt | 35 cycles |
| Cycle 2 | 40.5°C / 60°C | 25 sec | Anneal |  |
| Cycle 3 | 72°C | 30 sec per kb | Extend |  |
|  |  |  |  |  |
| Finish | 72°C | 5 min | Extend |  |
| Store | 10°C | Forever | Store |  |

**Digestion of SPCR 5 and SPCR7**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Purified PCR Product | 16 ul |
| H2O | 0 ul |
| 10x FastDigest Buffer | 2 ul |
| FastDigest Enzyme 1 | 1 ul |
| FastDigest Enzyme 2 | 1 ul |
| **Total Volume** | **20 ul** |

Incubate at 37°C for 1h shaking

**Purification of Digestion**
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 30 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Sunday 25th August

***Gel of Gradient PCR, Liquid Cultures, Ligation M13 + RFP, Ligation SPCR5, SPCR7, Electrocompetent cells, Gibson Assembly, Transformation (Gibson)and Transformation (Gibson, Ligations.***

**Gel of Gradient PCR**
1%, 100V, 20min

![](https://static.igem.org/mediawiki/2013/b/bd/13%EF%80%A208%EF%80%A225_SPCR2_iS001_iS002.jpg)

**Liquid Cultures**
NEB Turbo
XL10

**Ligation**

**Ligation M13 + RFP**
0.5ul Vector (M13)
2.6ul Insert (RFP)
5.4ul H20
1.0ul T4 buffer
0.5ul T4 Ligase

**Ligation SPCR5, SPCR7**
1.0ul Vector (SPCR5)
1.5ul Insert (SPCR7)
6.0ul H20
1.0ul T4 buffer
0.5ul T4 Ligase

Incubate ~30min at 37 C

**Electrocompetent cells**
1) Start Culture
2) When the cells reach an OD600 of 0.2.
3) Harvest the cells.
When the cells reach an OD600 of between 0.6 and 0.8 (OD: 0,683)
Centrifuge at 4 °C for 10 min at 4000 rpm.
4) Wash and combine the cells.
Remove the supernatant.
Resuspend the cells in 25 ml of ice cold water
5) Wash the cells 2 more times.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 50 ml of ice cold water.
Repeat.
6) Wash and concentrate the cells for electroporation.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 1-2 ml of ice cold water.
We will use 200 ul of washed cells per transformation.

**Gibson Assembly**

Amount per Reaction
Positive Control\*\*

PCR Fragment(s)
+ linearized vector
2-10 μl (0.02–0.5 pmols)\*

Gibson
Assembly Master
Mix (2X)
10 μl

Deionized H2O
XX μl

Total Volume
20 μl

Incubate samples in a thermocycler at 50°C for 15 minutes. After incubation, store the samples on ice or at -20°C for subsequent transformation
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.

**Transformation (Gibson)**
Thaw competent cells on ice.
Add 2 μl of the chilled assembly product to the competent cells. Mix gently by pipetting up and down or by flicking the tube 4–5 times. Do not vortex.
Place the mixture on ice for 30 minutes. Do not mix.
Heat shock at 42°C for 30 seconds. Do not mix.
Transfer tubes to ice for 2 minutes.
Add 950 μl of room-temperature SOC media to the tube.
Incubate the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
Warm selection plates to 37°C.
Spread 100 μl of the cells onto the selection plates. Use Amp plates for positive control sample.
Incubate overnight at 37°C.

**Transformation (Gibson, Ligations)**
1) Prepare BD tubes with a pipette filled with LB at the interior of each tube (pipette supplied with the electroporation cuvette)

2) Test the purity of the electrocompetent cells.
Add 200 ul of washed cells to a cuvette.

3) Mix the cells and DNA in a cuvette.
200 ul of washed cells with 200 ng of PCR product.
Keep the cuvette on ice until just before the electroporation.

4) Preload a pipette with 1 ml of LB.

5) Pulse the cuvette with voltage.
Dry the electrodes with a kimwipe prior to loading.
Use the EC2 setting.

6) Listen for arcing.
A cracking sound means all the cells are dead.
Note the time constant: 5 is good, 5.8 is great.

7) Immediately recover the cells.
Add the 1 ml of preloaded LB and pipet up and down to mix.
Collect 1 ml of cells, some volume is lost in the cuvette.

8) Incubate 2 h at 37 °C with shaking.

9) Plate 100 ul of recovered cells on selective plates.
Use antibiotic appropriate to the part being integrated.
Let the other 900 ul rest overnight at room temperature.

10) Concentrate and plate the remaining cells
Spin down quickly and resuspend in 100 ul LB before plating.

Transformed cells should be incubated at 37 °C.
Colonies should appear 24-48 h after plating.

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Monday 26th August

**Picking colonies (Trafo Gibson, BBC SPCR5/7)**
chosing 5-7 colonies and start liquid cultures

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Tuesday 27th August

***Starting liquid cultures and Miniprep of pS.005 and pS.006.***

**Starting liquid cultures**
sSP001, sSP002, sSP010, sSP011, sSP012, sSP020

**Miniprep of pS.005 and pS.006**
1) Pellet 2x 9ml of liquid culture (4000rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250ul of resuspension solution
4) add 250ul of lysis solution, mix by inverting 4-6 times
5) add 350ul of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 ul wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove left over liquid
10) transfer the column on a 1.5ml tube
11) add 50ul of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

**Colony PCR**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Forward Primer (10 uM) | 0,5 ul |
| Reverse Primer (10 uM) | 0,5 ul |
| Template DNA (Miniprep) | 0,5 ul |
| Quick-Load® Taq 2X Master Mix | 12,5 ul |
| Nuclease-free water | 10,0 ul |
| **Total volume** | 25 ul |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Thermocycler Protocol: Dream Taq Green** |  |  |  |  |
|  | Temp | Time |  |  |
| Start | 95°C | 30 sec1 | Melt |  |
|  |  |  |  |  |
| Cycle 1 | 95°C | 15 sec | Melt | 35 cycles |
| Cycle 2 | 46,8°C | 30 sec | Anneal |  |
| Cycle 3 | 68°C | 1 min per kb | Extend | cellule 2 |
|  |  |  |  |  |
| Finish | 68°C | 5 min | Extend |  |
| Store | 10°C | Forever | Store |  |

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Wednesday 28th August

***Gel Colony PCR, Liquid Cultures (1:100), Analytical Digest, Gel Ligation/Gibson, Gel Analytical digest, DpnI digest of SPCR5, SPCR6, SPCR7, Gel DpnI digest, PCR purification of DpnI digested SPCR5, SPCR6, SPCR7, Stress test microscope and M13 Test.***

**Gel Colony PCR**
pS005 (C1-C5) - pS006 (C1-C7)
1% 100V 20 min

![](https://static.igem.org/mediawiki/2013/b/b8/13%EF%80%A208%EF%80%A228_pS005_pS006_Colony_PCR_iS029_iS030.jpg)

**Liquid Cultures (1:100)**
sSP001, sSP002, sSP010, sSP011, sSP012, sSP020

**Analytical Digest**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Plasmid Miniprep | 5 ul |
| H2O | 12 ul |
| c10x FastDigest Buffer | 2ul |
| FastDigest Enzyme 1 | 0,5 ul |
| FastDigest Enzyme 2 | 0,5 ul |
| **Total Volume** | 20,0 ul |

Digest for 15 minutes at 37 °C with shaking

**Gel Ligation/Gibson**
pS005 - pS006 - M13/RFP
1% 100V 20min

![](https://static.igem.org/mediawiki/2013/7/7d/13%EF%80%A208%EF%80%A228_pS005_pS006_M13%EF%80%A2RFP_Ligation%EF%80%A2Gibson_products.jpg)

**Gel Analytical digest**
pS005 (C1-C5)-pS006 (C1-C7)
1% 100V 20min

![](https://static.igem.org/mediawiki/2013/1/1b/13%EF%80%A208%EF%80%A228_pS005_%28C1-C5%29_digested_with_XbaI%EF%80%A2SpeI_pS006_%28C1-C7%29_digested_with_XbaI%EF%80%A2SpeI.jpg)

**DpnI digest of SPCR5, SPCR6, SPCR7**
add 4ul of DpnI into each tube, mix and incubate for 1h at 37°C

**Gel DpnI digest**
let each 5ul run on a gel
1%, 100V 20min

**PCR purification of DpnI digested SPCR5, SPCR6, SPCR7**
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 30 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.

**Stress test microscope**
Dilute ON cultures of sSP001, sSP002, sSP010, sSP011, sSP012 1:100 and incubate up to an OD of 0.3
Take out 2ml of sSP001, sSP002, sSP010, sSP011
add:
sSP001: 10ug/ml MMC
sSP002: 10ug/ml MMC
sSP010: 20ul sSP012 supernatant
sSP011: 20ul sSP012 supernatant

incubate for 90 min

take pictures under the microscope (trans and YFP) of (un-)treated samples

**M13 Test**
Liquid cultures of sSP012 and sSP020
1) 100 μ l of plating bacteria per tube (OD 0.6)
2) Prepare tenfold serial dilutions (10-6 to 10-9 ) of the bacteriophage stock in LB
3) Put 10 μ l/ 100 μ l of each dilution to the bacteria
4) Mix the bacteriophage particles with the bacterial culture by vortexing gently.
5) Add 40 μ l of X-gal solution (working conc) and IPTG (working conc) solution to each of the tubes
6) Add 3ml of Agar to each tube and gently vortexing for 3 seconds
7) Pour the mixture onto plates containing LB medium supplemented with 5 mM MgCl2 and 2x IPTG and Xgal
8) Swirl the plate gently to ensure an even distribution of bacteria and agar.
9) Incubate them at 37°C.

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Friday 30th August

***M13 test, DpnI digest (SPCR5,6,7), Gel SPCR5,6,7 and PCR purification of DpnI digested SPCR5, SPCR6, SPCR7***

**M13 test**
dilute ON cultures (NEB turbo, FR-433) 1: 100, grow up to an OD of ~0.6
1) 100 μ l of plating bacteria per tube (OD 0.6)
2) Prepare tenfold serial dilutions (10-6 to 10-9 ) of the bacteriophage stock in LB
3) Put 10 μ l/ 100 μ l of each dilution to the bacteria
4) Mix the bacteriophage particles with the bacterial culture by vortexing gently.
5) Add 40 μ l of X-gal solution (working conc) and IPTG (working conc) solution to each of the tubes
6) Add 3ml of Agar to each tube and gently vortexing for 3 seconds
7) Pour the mixture onto plates containing LB medium supplemented with 5 mM MgCl2 and 2x IPTG and Xgal
8) Swirl the plate gently to ensure an even distribution of bacteria and agar.
9) Incubate them at 37°C.

**DpnI digest (SPCR5,6,7)**
add 8ul DpnI per tube and digest for 1h at 37°, shaking

**Gel SPCR5,6,7**
1%, 100V, 20min

![](https://static.igem.org/mediawiki/2013/7/72/13%EF%80%A208%EF%80%A230_sPCR5_SPCR6_sPCR7_after_DpnI_digest.jpg)

**PCR purification of DpnI digested SPCR5, SPCR6, SPCR7**
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 30 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Day Numbersuffix Month

***Place your twit here***

Place your note here

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Wednesday 4th September

***Starting Liquid culture, PCRs, Gel, DpnI digest, Digestion of SPCR5,7, Purification of Digestion, Ligation of SPCR5,7, Gibson Assembly, Making Electrocompetent Cells, Transformation, Redo SPCR12/13 ,***

**Starting Liquid culture**
NEB turbo, grow up to an OD of 0.6

**PCRs**
SPCR1, SPCR2, SPCR4, SPCR12, SPCR13

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **Reagant** | **Volume** |  |  |  |  |
|  | **1x (SPCR2)** | **11x (SPCR12)** | **11x (SPCR13)** | **8x (SPCR1)** | **8x (SPCR4)** |
| Nuclease-free water | 37.25 µl | 447 µl | 447 µl | 335,25µl | 335,25µl |
| 5x Phusion HF Buffer | 10 µl | 120 µl | 120 µl | 90 µl | 90 µl |
| 10 mM dNTPs | 1 µl | 12 µl | 12 µl | 9 µl | 9 µl |
| Forward Primer (10 uM) | 0.5 µl | 6 µl | 6 µl | 4.5 µl | 4.5 µl |
| Reverse Primer (10 uM) | 0.5 µl | 6 µl | 6 µl | 4.5 µl | 4.5 µl |
| Template Plasmid | 0.25 µl | 3 µl | 3 µl | 2.25 µl | 2.25 µl |
| Phusion DNA Polymerase | 0.5 µl | 6 µl | 6 µl | 4,5 µl | 4,5 µl |
| Total Volume | 50 µl | 600 µl | 600 µl | 450 µl | 450 µl |

for SPCR 12/13 gradient, 2,5 min
for SPCR3, SPCR11 51°, 45s
for SPCR 1, SPCR2, 48,8°, 2 min
for SPCR4, 40,8°, 1 min

**Gel**
1%, 100V, 20 min

1.small gel (SPCR1,2,3,4,11)
2. big gel (SPCR12)
3. big Gel (SPCR13)

![](https://static.igem.org/mediawiki/2013/b/b9/13%EF%80%A209%EF%80%A204_SPCR1%2C2%2C3%2C4%2C11_with_corresponding_primers.jpg)

![](https://static.igem.org/mediawiki/2013/1/13/13%EF%80%A209%EF%80%A204_SPCR12_gradient_wrong_programmed_corresponding_primers.jpg)

![](https://static.igem.org/mediawiki/2013/6/6b/13%EF%80%A209%EF%80%A204_SPCR13_gradient_wrong_programmed_corresponding_primers.jpg)

**DpnI digest**
SPCR 1: add 10ul DpnI
SPCR4: add 10ul DpnI
SPCR2,3,11: add 1 ul DpnI

incubate for 1h at 37°

**Digestion of SPCR5,7**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Purified PCR Product | 16 µl |
| H2O | 0 µl |
| 10x FastDigest Buffer | 2 µl |
| XbaI | 1 µl |
| SpeI | 1 µl |
| **Total Volume** | 20.0 µl |

Incubate for 1h at 37°, shaking

**Purification of Digestion**
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 30 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.

**Ligation of SPCR5,7**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Vector | 1 µl |
| Insert | 3 µl |
| H2O | 4.5 µl |
| Fermentas T4 Ligase Buffer | 1 µl |
| Fermentas T4 Ligase Enzyme | 0.5 µl |
| **Total Volume** | 10 µl |

Incubate at room temperature for ~1h

**Gibson Assembly**

**Amount per Reaction :** Positive Control\*\*

**PCR Fragment(s)+ linearized vector :** 2-10 μl (0.02–0.5 pmols)\*

**Gibson Assembly Master Mix (2X):** 10 μl

**Deionized H2O :** XX μl

**Total Volume :** 20 μl

Incubate samples in a thermocycler at 50°C for 15 minutes.
Put on Ice
Insert: 2,4 ul
Vector: 0,6 ul

**Making Electrocompetent Cells**

1) Start a liquid culture of cells.
NEB turbo
2) dilute the cells 100X.
100 ml LB.
Grow at 30°C for about 90 minutes.
3) Harvest the cells.
When the cells reach an OD600 of between 0.6 and 0.8
Split the culture into 2x 50 ml falcon tubes, on ice.
Centrifuge at 4 °C for 10 min at 4000 rpm.
5) Wash and combine the cells.
Remove the supernatant.
Resuspend the cells in 2x 25 ml of ice cold water.
Combine the volumes in a single 50 ml falcon tube.
6) Wash the cells 2 more times.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 50 ml of ice cold water.
Repeat.
7) Wash and concentrate the cells for electroporation.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 1-2 ml of ice cold water.
We will use 200 ul of washed cells per transformation.

**Transformation**
pS005, pS006, SPCR3 (IDT plasmid) M13mp18
1) Prepare BD tubes with a pipette filled with LB at the interior of each tube (pipette supplied with the electroporation cuvette)

2) Test the purity of the electrocompetent cells.
Add 200 ul of washed cells to a cuvette.

3) Mix the cells and DNA in a cuvette.
200 ul of washed cells with 200 ng of PCR product.
Keep the cuvette on ice until just before the electroporation.

4) Preload a pipette with 1 ml of LB.

5) Pulse the cuvette with voltage.
Dry the electrodes with a kimwipe prior to loading.
Use the EC2 setting.

6) Listen for arcing.
A cracking sound means all the cells are dead.
Note the time constant: 5 is good, 5.8 is great.

7) Immediately recover the cells.
Add the 1 ml of preloaded LB and pipet up and down to mix.
Collect 1 ml of cells, some volume is lost in the cuvette.

8) Incubate 2 h at 37 °C with shaking.

9) Plate 10/200 ul of recovered cells on selective plates.
Use antibiotic appropriate to the part being integrated.
Let the other 900 ul rest overnight at room temperature.

10) Concentrate and plate the remaining cells
Spin down quickly and resuspend in 100 ul LB before plating.

Transformed cells should be incubated at 37 °C.
Colonies should appear 24-48 h after plating.

**Redo SPCR12/13**

|  |  |  |
| --- | --- | --- |
| **Reagent** | **Volume** |  |
|  | **11x (SPCR12)** | **11x (SPCR13)** |
| Nuclease-free water | 447 µl | 447 µl |
| 5x Phusion HF Buffer | 120 µl | 120 µl |
| 10 mM dNTPs | 12 µl | 12 µl |
| Forward Primer (10 uM) | 6 µl | 6 µl |
| Reverse Primer (10 uM) | 6 µl | 6 µl |
| Template Plasmid | 3 µl | 3 µl |
| Phusion DNA Polymerase | 6 µl | 6 µl |
| **Total Volume** | 600 µl | 600 µl |

Cycling protocol as usual + gradient + topdown

![](https://static.igem.org/mediawiki/2013/b/b6/Cycler_protocol_4.9.13.JPG)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Thursday 5th September

***Gel, Colony PCR, Topdown PCR and gels***

**Gel**
SPCR12, SPCR13
1%, 100V, 20 min

Pictures: 13/09/05\_SPCR12\_corresponding primers
![](https://static.igem.org/mediawiki/2013/7/72/3-09-05_SPCR12_corresponding_primers.png)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Friday 6th September

***Place your twit here***

Digestions: linearized pSB1C3, linearized pSB1A3, SPCR2, SPCR3, SPCR11

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Purified PCR Product | 16 µl |
| H2OH2O | 0 µl |
| 10x FastDigest Buffer | 2 µl |
| FastDigest Enzyme 1 | 1 µl |
| cFastDigest Enzyme 2 | 1 µl |
| **Total Volume** | 4 µl |

SPCR5, pSB1C3: XbaI/SpeI
SPCR11, pSB1A3, pSB1C3: EcoRI/PstI
SPCR13, pSB1A3, pSB1C3: EcoRI/PstI
SPCR2, pSB1A3, pSB1C3: EcoRI/PstI

1h at 37° shaking

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Saturday 7th September

***Ligation, Making Electrocompetent Cells, Gibson Assembly, Transformation, Liquid culture of E1010***

**Ligation**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Vector | 1 µl |
| Insert | 3 µl |
| H2O | 4.5 µl |
| Fermentas T4 Ligase Buffer | 1 |
| Fermentas T4 Ligase Enzyme | 0.5 |
| **Total Volume** | 10 µl |

SPCR5, pSB1C3
SPCR11, pSB1A3, pSB1C3
SPCR13, pSB1A3, pSB1C3
SPCR2, pSB1A3, pSB1C3

Incubate at room temperature for 10 minutes.

**Making Electrocompetent Cells**
1) Start a liquid culture of cells.
NEB turbo
2) dilute the cells 100X.
100 ml LB.
Grow at 30°C for about 90 minutes.
3) Harvest the cells.
When the cells reach an OD600 of between 0.6 and 0.8
Split the culture into 2x 50 ml falcon tubes, on ice.
Centrifuge at 4 °C for 10 min at 4000 rpm.
5) Wash and combine the cells.
Remove the supernatant.
Resuspend the cells in 2x 25 ml of ice cold water.
Combine the volumes in a single 50 ml falcon tube.
6) Wash the cells 2 more times.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 50 ml of ice cold water.
Repeat.
7) Wash and concentrate the cells for electroporation.
Centrifuge at 4 °C for 10 min at 4000 rpm.
Resuspend in 1-2 ml of ice cold water.
We will use 200 ul of washed cells per transformation.

**Gibson Assembly**

Amount per Reaction
Positive Control\*\*

PCR Fragment(s)
+ linearized vector
2-10 μl (0.02–0.5 pmols)\*

Gibson
Assembly Master
Mix (2X)
10 μl

Deionized H2O
XX μl

Total Volume
20 μl

Incubate samples in a thermocycler at 50°C for 15 minutes.
Put on Ice

**Transformation**
pS002 (Chl), pS002 (Amp), pS003 (Amp), pS004 (Chl), pS004 (Amp), pS005 (Chl), pS006-7 (Chl), pS007 (Chl), pS008 (Amp), pS009 (Chl), pS012 (Amp), pS013 (Chl), pS013 (Amp)

1) Prepare BD tubes with a pipette filled with LB at the interior of each tube (pipette supplied with the electroporation cuvette)

2) Test the purity of the electrocompetent cells.
Add 200 ul of washed cells to a cuvette.

3) Mix the cells and DNA in a cuvette.
200 ul of washed cells with 200 ng of PCR product.
Keep the cuvette on ice until just before the electroporation.

4) Preload a pipette with 1 ml of LB.

5) Pulse the cuvette with voltage.
Dry the electrodes with a kimwipe prior to loading.
Use the EC2 setting.

6) Listen for arcing.
A cracking sound means all the cells are dead.
Note the time constant: 5 is good, 5.8 is great.

7) Immediately recover the cells.
Add the 1 ml of preloaded LB and pipet up and down to mix.
Collect 1 ml of cells, some volume is lost in the cuvette.

8) Incubate 1/2 h at 37 °C with shaking.

9) Plate 10/200 ul of recovered cells on selective plates.
Use antibiotic appropriate to the part being integrated.
Let the other 900 ul rest overnight at room temperature.

10) Concentrate and plate the remaining cells
Spin down quickly and resuspend in 100 ul LB before plating.

Transformed cells should be incubated at 37 °C.
Colonies should appear 24-48 h after plating.

**Liquid culture of E1010**

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Sunday 8th September

***Replating of transformation and Miniprep of E1010***

**Replating of Transformation**

**Miniprep of E1010**
1) Pellet liquid culture (4000rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250ul of resuspension olution
4) add 250ul of lysis solution, mix by inverting 4-6 times
5) add 350ul of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 ul wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove left over liquid
10) transfer the column on a 1.5ml tube
11) add 50ul of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Monday 16th September

***Result Cloning, Protocol: E. coli Colony PCR, Extraction of DNA, PCR Reaction and Gels***

**Result Cloning:**
Colonies?
pS002 (Chl): no
pS002 (Amp): yes
pS003 (Amp):yes
pS004 (Chl): no
pS004 (Amp): yes
pS005 (Chl): yes
pS006-7 (Chl): yes
pS006 (Chl): no
pS007 (Chl): yes
pS008 (Amp): yes
pS009 (Chl): yes
pS012 (Amp): yes
pS013 (Chl): no
pS013 (Amp): yes

**Protocol: E. coli Colony PCR**
Using the Dream Taq Master Mix

**Extraction of DNA**

1) Pick a single colony into 50 ul of H20.

2) Boil for 5 minutes.

**PCR Reaction (pS002 (Chl-Lig), pS005 (Chl-G), pS006-7 (Chl-Lig), pS007 (Chl-G), pS008 (Amp-G), pS009 (Chl-G))**

Keep all the reagents at 4 °C while preparing the mixture.
Pre-heat the thermocycler to 95 °C and transfer your reaction directly from 4 °C.

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Forward Primer (10 uM) | 0.5 ul |
| Reverse Primer (10 uM) | 0.5 ul |
| Template DNA (from above) | 1.5 ul |
| Quick-Load® Taq 2X Master Mix | 12.5 ul |
| Nuclease-free water | 10 ul |
| **Total Volume** | 25 ul |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Thermocycler Protocol: NEB Quick-Load** |  |  |  |  |
|  | Temp | Time |  |  |
| Start | 95°C | 30 sec | Melt |  |
|  |  |  |  |  |
| Cycle 1 | 95°C | 15 sec | Melt | 35 cycles |
| Cycle 2 | 45.8 °C | 30 sec | Anneal |  |
| Cycle 3 | 72°C | 1 min per kb | Extend |  |
|  |  |  |  |  |
| Finish | 72 °C | 15 min | Extand |  |
| Store | 10°C | Forever | Store |  |

**Gels**
100V, 25-30 min, 1%

Pictures:
13/09/16\_colony PCR\_ps007.BMP
![](https://static.igem.org/mediawiki/2013/5/50/13-09-16_colony_PCR_ps007.png)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Tuesday 17th September

***PCR Reaction (pS003 (Amp-G), pS004 (Amp-Lig), pS012 (Amp-G), pS013 (Chl-Lig))***

**PCR Reaction (pS003 (Amp-G), pS004 (Amp-Lig), pS012 (Amp-G), pS013 (Chl-Lig))**

Keep all the reagents at 4 °C while preparing the mixture.
Pre-heat the thermocycler to 95 °C and transfer your reaction directly from 4 °C.

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Forward Primer (10 uM) | 0.5 ul |
| Reverse Primer (10 uM) | 0.5 ul |
| Template DNA (from above) | 1.5 ul |
| Quick-Load® Taq 2X Master Mix | 12.5 ul |
| Nuclease-free water | 10 ul |
| **Total Volume** | 25 ul |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Thermocycler Protocol: NEB Quick-Load** |  |  |  |  |
|  | Temp | Time |  |  |
| Start | 95°C | 30 sec | Melt |  |
|  |  |  |  |  |
| Cycle 1 | 95°C | 15 sec | Melt | 35 cycles |
| Cycle 2 | 45.8 °C | 30 sec | Anneal |  |
| Cycle 3 | 72°C | 1 min per kb | Extend |  |
|  |  |  |  |  |
| Finish | 72 °C | 15 min | Extand |  |
| Store | 10°C | Forever | Store |  |

**Gels**
100V, 30 min, 1%

**Pictures:**
13/09/17\_colony PCR\_pS009.JPG
![](https://static.igem.org/mediawiki/2013/b/b5/13-09-17_colony_PCR_pS013.JPG)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Wednesday 16th September

***Ligations***

**Ligation**

SPCR2 - Chl

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| SPCR2 | 1 µl |
| Chl | 2,95 µl |
| H2O | 4.55 µl |
| Fermentas T4 Ligase Buffer | 1.0 µl |
| Fermentas T4 Ligase Enzyme | 0.5 µl |
| **Total Volume** | 10 µl |

SPCR2 - Amp

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| SPCR2 | 1 µl |
| Amp | 3.4 µl |
| H2O | 4.1 µl |
| Fermentas T4 Ligase Buffer | 1.0 µl |
| Fermentas T4 Ligase Enzyme | 0.5 µl |
| **Total Volume** | 10 µl |

SPCR3 - Chl

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| SPCR3 | 1 µl |
| Amp | 1.79 µl |
| H2O | 5.71 µl |
| Fermentas T4 Ligase Buffer | 1.0 µl |
| Fermentas T4 Ligase Enzyme | 0.5 µl |
| **Total Volume** | 10 µl |

SPCR3 - Amp7

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Amp | 1 µl |
| SPCR 3 | 1.56 µl |
| H2O | 5.94 µl |
| Fermentas T4 Ligase Buffer | 1.0 µl |
| Fermentas T4 Ligase Enzyme | 0.5 µl |
| **Total Volume** | 10 µl |

SPCR5 - Chl

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| SPCR 5 | 0.5 µl |
| Chl | 4.55 µl |
| H2O | 3.45 µl |
| Fermentas T4 Ligase Buffer | 1.0 µl |
| Fermentas T4 Ligase Enzyme | 0.5 µl |
| **Total Volume** | 10 µl |

SPCR11 - Chl

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Chl | 1 µl |
| SPCR11 | 1.3 µl |
| H2O | 6.2 µl |
| Fermentas T4 Ligase Buffer | 1.0 µl |
| Fermentas T4 Ligase Enzyme | 0.5 µl |
| **Total Volume** | 10 µl |

SPCR2 - Amp

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Amp | 1 µl |
| SPCR11 | 1.13 µl |
| H2O | 6.37 µl |
| Fermentas T4 Ligase Buffer | 1.0 µl |
| Fermentas T4 Ligase Enzyme | 0.5 µl |
| **Total Volume** | 10 µl |

at 16° for 1h

**Transformation**
1) Thaw competent cells on ice (Gibson Kit competent cells)
2) Place 20 ul of cells in a pre-chilled Eppendorf tube.
3) Add 2.5 ul of plasmid (from Biobrick stock)
4) Mix gently by flicking the tube.
5) Chill on ice for 10 minutes.
4) Heat shock at 42 °C for 30 seconds
5) Return to ice for 2 minutes.
6) Add 200 ul LB medium and recover the cells by shaking at 37 °C.
Ampicillin: 15-30 minutes
Chloramphenicol: 60-120 minutes
7) Plate out the cells on selective LB (10ul)
8. Incubate at 37 °C. Transformants should appear within 12 hrs.

**Gel of Ligation products**
1%, 100V, 40 min

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Sunday 22nd September

***PCR and Gels***

**PCR**
M13 backbone

SPCR16,17

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
|  | **x1** |
| Nuclease-free water | 37.25 µl |
| 5x Phusion HF Buffer | 10 µl |
| 10 mM dNTPs | 1 µl |
| Forward Primer (10 uM) | 0.5 µl |
| Reverse Primer (10 uM) | 0.5 µl |
| Template Plasmid | 0.25 µl |
| Phusion DNA Polymerase | 0.5 µl |
| **Total Volume** | 50 µl |

Protocol see fotos

**Gel**
100V, 1%, 1,5h

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Monday 23rd September

***Gels and PCR***

**Gel**
100V, 1%, 1,5h

Picture: 13/09/23\_SPCR16\_17\_corresponding primers - PCRs didn't worked
![](https://static.igem.org/mediawiki/2013/0/0e/13-09-23_SPCR16_17_corresponding_primers.JPG)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Tuesday 24th September

***Overlap PCR, PCR of Overlap, Digestion, Gel extraction, Gels, PCR Purification, PCR (SPCR2, SPCR3, SPCR5, SPCR6, SPCR7, SPCR8, SPCR9, SPCR10, SPCR11, linearize pSB1C3) and liquid culture***

**Overlap PCR**
SPCR5-SPCR2

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
|  | **x1** |
| Nuclease-free water | 37.25 µl |
| 5x Phusion HF Buffer | 10 µl |
| 10 mM dNTPs | 1 µl |
| Template Plasmid(10 uM) | 1 µl |
| Template Plasmid (10 uM) | 1 µl |
| Phusion DNA Polymerase | 0.5 µl |
| **Total Volume** | 50 µl |

![](https://static.igem.org/mediawiki/2013/8/8b/PB_labbooksensor01.JPG)
![](https://static.igem.org/mediawiki/2013/6/6b/PB_labbooksensor02.JPG)

**PCR of Overlap**

**Digestion**
M13 (EcoRI, PstI), pSB1C3 (EcoRI, PstI), psB1C3 (XbaI, SpeI)

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Purified Plasmid | 20 µl |
| H2O | 63 µl |
| 10x FastDigest Buffer | 10 µl |
| FastDigest Enzyme 1 | 2.5 µl |
| FastDigest Enzyme 2 | 2.5 µl |
| FastAP Phosphatase | 2 µl |
| **Total Volume** | 100 µl |

SPCR2 (EcoRI, PstI), SPCR5 (XbaI, SpeI)

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Purified PCR Product | 16 µl |
| H2O | 0 µl |
| 10x FastDigest Buffer | 2 µl |
| FastDigest Enzyme 1 | 1 µl |
| FastDigest Enzyme 2 | 1 µl |
 **Total Volume** | 20 µl |

**Gel**
pSB1C3
1%, 100V, 1h
Picture: 13/09/24\_pSB1C3 digest\_EcoRI/PstI\_XbaI/SpeI - digest worked
![](https://static.igem.org/mediawiki/2013/e/ec/13-09-24_pSB1C3_digest_EcoRI-PstI_XbaI-SpeI.JPG)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Wednesday 25th September

***Gel of PCR, PCR (SPCR5,6) and Miniprep Cas9, M13mp18***

**Gel of PCR**
1%, 100V, 1h

Picture: 13/09/25\_SPCR2-11 - SPCR2: worked, SPCR3: worked, SPCR5: didn't work, SPCR6: didn't work, SPCR7: didn't work, SPCR8: didn't work, SPCR9: didn't work, SPCR10: didn't work, SCPR11: worked
![](https://static.igem.org/mediawiki/2013/c/ca/13-09-25_SPCR2-10.png)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Thursday 26th September

***Gel PCR 5,6, Digestion, PCR (SPCR7-10) and PCR purification***

**Gel PCR 5,6**
1%, 100V, 1h

Picture: 13/09/26\_SPCR5\_6 - PCR didn't work
![](https://static.igem.org/mediawiki/2013/b/b2/13-09-26_SPCR5_6.png)

**Digestions: SPCR2 (EcoRI, PstI), SPCR3 (EcoRI, PstI), SPCR4 (EcoRI, PstI), SPCR11 (EcoRI, PstI), lin pSB1C3 (EcoRI, PstI), lin pSB1A3 (EcoRI, PstI), SPCR5 (XbaI, SpeI), SPCR7 (XbaI, SpeI), lin pSB1C3 (XbaI, SpeI), in pSB1A3 (XbaI, SpeI)**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
| Purified PCR Product | 16 µl |
| H2O | 0 µl |
| 10x FastDigest Buffer | 2 µl |
| FastDigest Enzyme 1 | 1 µl |
| FastDigest Enzyme 2 | 1 µl |
| **Total Volume** | 20 µl |

**PCR (SPCR7-10)**

|  |  |
| --- | --- |
| **Reagent** | **Volume** |
|  | **x1** |
| Nuclease-free water | 37.25 µl |
| 5x Phusion HF Buffer | 10 µl |
| 10 mM dNTPs | 1 µl |
| Forward Primer (10 uM) | 1 µl |
| Reverse Primer (10 uM) | 1 µl |
| Template Plasmid) | 0.25 µl |
| Phusion DNA Polymerase | 0.5 µl |
| **Total Volume** | 50 µl |

**Thermocycler Protocol: Fermentas Phusion** |  |  |  |  ||  | Temp | Time |  |  |
| Start | 98°C | 30 sec | Melt |  |
|  |  |  |  |  |
| Cycle 1 | 98°C | 5 sec | Melt | 35 cycles |
| Cycle 2 |  | 25 sec | Anneal |  |
| Cycle 3 | 72°C | 30 sec per kb | Extend |  |
|  |  |  |  |  |
| Finish | 72 °C | 5 min | Extand |  |
| Store | 10°C | Forever | Store |  |

**PCR Purification: (SPCR2 (EcoRI, PstI), SPCR3 (EcoRI, PstI), SPCR4 (EcoRI, PstI), SPCR11 (EcoRI, PstI), lin pSB1C3 (EcoRI, PstI), lin pSB1A3 (EcoRI, PstI), SPCR5 (XbaI, SpeI), SPCR7 (XbaI, SpeI), lin pSB1C3 (XbaI, SpeI), in pSB1A3 (XbaI, SpeI)**

Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 μl Buffer EB to the center of the QIAquick membrane and centrifuge the column for 1 min.

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Friday 27th September

***Gel SPCR7-10, NEB liquid culture, Ligation, Electrocompetent cells and transformation***

**Gel SPCR7-10**
1%, 100V, 1h

Picture: 13/09/27\_SPCR7\_8\_9\_10 - PCRs worked
![](https://static.igem.org/mediawiki/2013/9/91/13-09-27_SPCR7_8_9_10.png)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Saturday 28th September

***Extraction of Genomic DNA, PCR Reaction and Restreak plates***

**Protocol: E. coli Colony PCR (pS006’, pS006\*, pS013, pS004, pS004\*, pS002\*)**

**Extraction of Genomic DNA**

1) Pick a single colony into 50 ul of H20.
Fresh colonies (grown that day) work best, but they can also come from 4 C.
Pipet 2ul onto plates to have C1-C8
Pipet 2 ul into 5ml Media with antibiotics to make liquid cultures

2) Boil for 5 minutes.
1.5 ul of this can be used directly for PCR.
Best if used directly, but can also be stored at 4 C for a few days.

**PCR Reaction**

Keep all the reagents at 4 °C while preparing the mixture.
Pre-heat the thermocycler to 95 °C and transfer your reaction directly from 4 °C.

|  |  |  |
| --- | --- | --- |
| **Reagent** | **Volume** | **9x Volume** |
| Forward Primer (10 uM) | 0.5 µl | 4.5 µl |
| Reverse Primer (10 uM) | 0.5 µl | 4.5 µl |
| Template DNA (from above) | 1.5 µl | 13.5 µl |
| Quick-Load® Taq 2X Master Mix | 12.5 µl | 112.5 µl9x Volume |
| Nuclease-free water | 10 µl | 90 µl |
| **Total Volume** | 25 µl | 125 µl |

**Thermocycler Protocol: Green Dream Taq** |  |  |  |  ||  | Temp | Time |  |  |
| Start | 95°C | 30 sec | Melt |  |
|  |  |  |  |  |
| Cycle 1 | 95°C | 15 sec | Melt | 35 cycles |
| Cycle 2 | 46.8°C | 30 sec | Anneal |  |
| Cycle 3 | 72°C | 1 min per kb | Extend |  |
|  |  |  |  |  |
| Finish | 72 °C | 10 min | Extand |  |
| Store | 10°C | Forever | Store |  |

**Restreak plates**
Some of the plates were too full grown -> Rstreak for single colonies

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Monday 30th September

***Extraction of Genomic DNA, PCR reaction, Gels, Miniprep, PCR: SPCR5, SPCR6 and Liquid culture of pS004’ and pS013’***

**Protocol: E. coli Colony PCR (pS006, pS013’,pS013\*, pS004’, pS002’)**

**Extraction of Genomic DNA**

1) Pick a single colony into 50 ul of H20.
Fresh colonies (grown that day) work best, but they can also come from 4 C.
Pipet 2ul onto plates to have C1-C8

2) Boil for 5 minutes.
1.5 ul of this can be used directly for PCR.
Best if used directly, but can also be stored at 4 C for a few days.

**PCR Reaction**

Keep all the reagents at 4 °C while preparing the mixture.
Pre-heat the thermocycler to 95 °C and transfer your reaction directly from 4 °C.

|  |  |  |
| --- | --- | --- |
| **Reagent** | **Volume** | **9x Volume** |
| Forward Primer (10 uM) | 0.5 µl | 4.5 µl |
| Reverse Primer (10 uM) | 0.5 µl | 4.5 µl |
| Template DNA (from above) | 1.5 µl | 13.5 µl |
| Quick-Load® Taq 2X Master Mix | 12.5 µl | 112.5 µl9x Volume |
| Nuclease-free water | 10 µl | 90 µl |
| **Total Volume** | 25 µl | 125 µl |

**Thermocycler Protocol: Green Dream Taq** |  |  |  |  ||  | Temp | Time |  |  |
| Start | 95°C | 30 sec | Melt |  |
|  |  |  |  |  |
| Cycle 1 | 95°C | 15 sec | Melt | 35 cycles |
| Cycle 2 | 46.8°C | 30 sec | Anneal |  |
| Cycle 3 | 72°C | 1 min per kb | Extend |  |
|  |  |  |  |  |
| Finish | 72 °C | 10 min | Extand |  |
| Store | 10°C | Forever | Store |  |

**Gels**
1%, 100V, 45 min

Pictures + Results
13/09/30\_colony pcr\_pS004\_pS013 - 1-8: pS004 (gRNA - Amp), 9-16: pS013 (crRNA - Amp) - seems to have worked beside pS013 C7
![](https://static.igem.org/mediawiki/2013/c/c5/13-09-30_colony_pcr_pS004_pS013.png)

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Tuesday 1st October

***Miniprep
pS013’, pS004’***

1) Pellet liquid culture (4000rpm, 10 min)
2) Discard supernatant
3) resuspend the cells in 250ul of resuspension olution
4) add 250ul of lysis solution, mix by inverting 4-6 times
5) add 350ul of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 ul wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove left over liquid
10) transfer the column on a 1.5ml tube
11) add 50ul of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Wednesday 2nd October

***Preparation killing assay***

**Liquid cultures**
R16, R26, R26f, delta PyrF, parental delta PyrF, NEB, M13, Litmus + Helper, Litmus GFP + helper

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Wedsnesday 3rd October

***Killing assay to test specifity of CRISPR Cas***

**Electrocompetent cells (delta PyrF, parental, NEB)**

1) Start Culture

2) When the cells reach an OD600 of 0.2.

3) Harvest the cells.

When the cells reach an OD600 of between 0.6 and 0.8 (OD: 0,683)

Centrifuge at 4 °C for 10 min at 4000 rpm.

4) Wash and combine the cells.

Remove the supernatant.

Resuspend the cells in 25 ml of ice cold water

5) Wash the cells 2 more times.

Centrifuge at 4 °C for 10 min at 4000 rpm.

Resuspend in 50 ml of ice cold water.

Repeat.

6) Wash and concentrate the cells for electroporation.

Centrifuge at 4 °C for 10 min at 4000 rpm.

Resuspend in 1-2 ml of ice cold water.

We will use 200 ul of washed cells per transformation.

**Transformation**

Cotransform into parental/keio delta pyRF: pS006\* (Cas9 in Amp BB) + pS004'(gRNA in Chl BB).
1) Prepare BD tubes with a pipette filled with LB at the interior of each tube (pipette supplied with the electroporation cuvette)

2) Test the purity of the electrocompetent cells.

Add 200 ul of washed cells to a cuvette.

3) Mix the cells and DNA in a cuvette.

200 ul of washed cells with 200 ng of PCR product.

Keep the cuvette on ice until just before the electroporation.

4) Preload a pipette with 1 ml of LB.

5) Pulse the cuvette with voltage.

Dry the electrodes with a kimwipe prior to loading.

Use the EC2 setting.

6) Listen for arcing.

A cracking sound means all the cells are dead.

Note the time constant: 5 is good, 5.8 is great.

7) Immediately recover the cells.

Add the 1 ml of preloaded LB and pipet up and down to mix.

Collect 1 ml of cells, some volume is lost in the cuvette.

8) Incubate 1/2 h at 37 °C with shaking.

9) Plate 10/200 ul of recovered cells on selective plates.

Use antibiotic appropriate to the part being integrated.

Let the other 900 ul rest overnight at room temperature.

10) Concentrate and plate the remaining cells

Spin down quickly and resuspend in 100 ul LB before plating.

Transformed cells should be incubated at 37 °C.

Colonies should appear 24-48 h after plating.

Plating of 10/100ul on
1. Amp plates -> selection for only Cas9
2. Chl plates -> selection for only gRNA
3. Amp/Chl plates -> selection for theoretically functional system

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Monday 14th October

***Liquid cultures sSP008, sSP009, R16, R26, R26f, sT007, sSP021, SCPR2-A3, SPCE2-C3***

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Tuesday 15th October

***Digestion Reporter and SPCR3***

BB: SpeI, PstI
SPCR: XbaI, PstI
2h 37°, heat inactivation

Electrocompetent cells: sSP008, sSP009
Transform SPCR2-A3/C3 both into sSP008/9

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Wednesday 16th October

***Miniprep of pS002, pS002’, pS006\*
Liquid culture sSP017, pS004, pS004’
Glycerols***

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Thursday 17th October

***Minipreps, Digestions, Ligation and Transformation***

Miniprep sSP017, pS004, pS004’

Digest Litmus pS018 EcoRI/PstI, EcoRI/XbaI 2h 37° shaking

PCR purify

Ligation:
1. gRNA+phagmid (10,4+6)
2.phagemid+gRNA+reporter (9,6+1,86+2)
3.phagemid+gRNA+Cas9 (10,2+2,04+3)

1h at 22°

Transform
Heat shock, cells matt
Recover, plate

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Friday 18th October

***Miller assay and Liquid phagemid***

 **Miller assay**
- Grow liquid cultures up to an OD 600 of 0.3-0.7
- Meassure the OD 600
- Start the specific cultures you want to test under the specific conditions
- Take each 600 ul of culture and add 400ul of Z buffer (see below)
- Add 100ul of Chloroform and 50ul of 0.1% SDS and vortex intensively
- Incubate the tubes for 5min at 28°C
- Add 200ul of ONPG (see below) and note the time point of adding
- Incubate for up to 90 min or until a yellow/green color appears
- To stop the reaction, add 500ul of Na2CO3 and note the time point of stopping
- Centrifuge for 3 min
- Measure the OD at 420

The expression of beta-galactosidase can now be calculated with the formula: Miller unit= (1000\*OD420)/(delta t\*V(culture taken)\*OD600)

Z-buffer:

- Na2HPO4 7H2O 60 mM - 16.1g
- NaH4PO4 H2O 40 mM - 5.5g
- KCl 10 mM - 0.75g
- MgSO4 7H2O 1 mM - 0.246g
- beta mercaptoethanol 50 mM - 2.7 ml

ONPG:

4mg/ml in Z-buffer

Na2CO3 1M

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Saturday 19th October

***Miller assay, Digestions***

**Miller
Miniprep phagemid
Digest phagemid E/P, E/X, SPCR3 E,P**

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Tuesday 22nd October

***Liquid for Miller and Digest&purify SPCR3 E,P***

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Thursday 24th October

***Digestion, Ligation, Transformation Ligation, Transformation***

**Digest phagemid E/P**
Purify it

**Ligation:**
1. SPCR3 E/P + phagemid E/P
2. SPCR3 E/P + SPCR5 P/X+ phagemid E/X
2. SPCR3 E/P + SPCR2 P/X+ phagemid E/X

**trafo Ligations

electrocmpetent cells parental+sSP009+pS002’
trafo:**
1. parental+pS002
2. parental+pS002’
3. parental+pS006\*
4. parental+pS002’+pS006\*
5. sSP009 pS002’ + pS006\*

trafo helper into NEB

liquid cultures: sSP009, pRECA strains Ariel, sSP008+pS002, sSP008+pS002’

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Friday 25th October

***Liquid cultures, Transformation, Conjugation***

**Colony PCR
Liquid Cloning**

Liquid: NEB helper, parental pS002, parental pS006\*, sSP008+pS002, sSP008+pS002’

Miniprep phagemid gRNA

Trafo: NEB helper+phageid-gRNA, parental ps006\*+pS002’, pyrfF F+ pS002’+pS006\*

conjugation of parental pS006\* with pS009

Liquid library

Restreak for library

 **Functionality Test of reporter**
1) sSP008+pS002/pS002’ +Xgal
2) sSP008+pS002/pS002’ +Xgal + MMC
3) sSP008+pS002/pS002’ +MMC

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Saturday 26th October

***Liquid cultures, Transformation, Conjugation***

**Liquid cultures**
sSP008, transformed parental strains, Kan strains, NEB helper-gRNA, sSP009

Conjugate at OD 0.2 parental + pS002'+pS006\* with sSP009 and plate 50ul on Amp/Tet/Chl plates

 **Electrocompetent cells+ transformation**

Trafo:sSP008+Cas9 Addgene+ phagemid-gRNA -> killing assay to confirm gRNA on phagemid works , pyrfF F+ pS002’+pS006\*

## Detect

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Detect)

### Sunday 27th October

***Miller assay final strain/construct with controls***

**Miller assay**
- MG1655 as positive control
- parental + reporter + Cas9 + F+
- Keio delta pyrF + reporter + Cas9 + F+
- parental + reporter + Cas9 + F+ + phagemid with gRNA
- Keio delta pyrF + reporter + Cas9 + F+ + phagemid with gRNA
- Keio delta pyrF + reporter + MMC
- parental + reporter + MMC

**Liquid culture functionality test**
- parental + reporter + Cas9 + F+ + Xgal
- parental + reporter + Cas9 + F+ + Xgal + phagemid with gRNA
- parental + reporter + Cas9 + F+ + phagemid with gRNA
- Keio delta pyrF + reporter + Cas9 + F+ + Xgal
- Keio delta pyrF + reporter + Cas9 + F+ + Xgal + phagemid with gRNA
- Keio delta pyrF + reporter + Cas9 + F+ + phagemid with gRNA

**Killing assay**
Plate:- parental + reporter + Cas9 + F+
- Keio delta pyrF + reporter + Cas9 + F+
- parental + reporter + Cas9 + F+ + phagemid with gRNA
- Keio delta pyrF + reporter + Cas9 + F+ + phagemid with gRNA

## Target

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Target)

### Monday 10th June

***Design of SirA genes for synthesis***

**SirA**

*Mycobacterium tuberculosis*

Reference: Pinto et al. 2007. Sulfite Reduction in Mycobacteria doi: 10.1128/JB.00487-07

Primers:

TBsirA F      GGAATTCCATATGTCCGCGAAGGAGAACCCC

TBsirA R      AAGGAAAAAAGCGGCCGCTCATCGCAGGTCGTCCTCCTCGGCCCGGAT
We blasted the above primers used to clone sirA from M. tuberculosis into E. coli. We then extracted the sequence from genbank to retrieve the following sequence which includes the restriction sites introduced by Pinto et al.

ggaattccat atgtccgcga aggagaaccc ccaaatgacc actgcacgtc
ccgccaaggc tcgaaatgag
ggccagtggg cgctgggaca tcgcgagcca
ctcaacgcca acgaagagct gaagaaggcc
ggcaacccgc tcgacgtgcg
ggagcgcatc gaaaacatct acgccaaaca gggtttcgac
agcatcgaca
agaccgacct gcgagggcgc tttcgctggt ggggcctgta cacccagcgt

gagcagggct acgacggcac ctggaccggt gacgacaaca tcgacaagct
cgaggccaaa
tacttcatga tgcgggtgcg ttgcgacggc ggcgcgctct
cggctgccgc gctgcgcacg
ctgggccaga tctcgacgga gttcgcgcgc
gataccgccg atatctccga ccggcagaac
gtgcaatacc actggatcga
agtggaaaac gtccctgaaa tctggcgacg gttagacgat
gtcggactgc
agaccaccga ggcgtgcggt gactgcccgc gggtagtgct gggctcgccg

ttggccggcg agtcgctcga cgaagtgctc gacccgacct gggcgatcga
ggagatcgtg
cgtcgctaca tcggcaagcc cgacttcgcc gacttgccgc
gcaagtacaa gaccgccatc
tctggcctgc aggacgtcgc gcacgagatc
aacgacgtcg ccttcatcgg cgtcaaccat
cccgagcacg gaccaggcct
ggatctgtgg gtgggcggtg gactgtcgac caacccgatg
ctggcccagc
gggtcggcgc ctgggttcca ctgggcgaag tgcccgaggt gtgggcggcg

gtcacctcgg tgtttcgcga ctacggctac cggcgactgc gcgccaaggc
ccggctgaaa
tttctgatca aagactgggg catagcgaag ttccgcgaag
tgctcgaaac cgagtacctc
aagcgtccgc tgatcgacgg tccggccccc
gaaccggtca agcatccgat cgaccacgtc
ggggtgcaac gactcaagaa
cgggctcaac gccgtcggag tcgcccccat cgccgggcgg
gtatcgggca
ccatcctcac ggcggtcgcc gacctgatgg cgcgggccgg ttccgaccgg

atccggttca ccccctacca gaagctggtc atcctcgaca ttccggacgc
cttgctcgac
gacttgatcg ccggtctgga cgcgctgggg ctgcagtcgc
gcccgtcgca ttggcgccgg
aacttgatgg cgtgcagcgg gattgagttc
tgcaagttgt cattcgccga aacccgggtt
cgagcacagc atttggtgcc
cgagctggaa cgccggcttg aggacatcaa ctcgcagctc
gacgtaccga
tcaccgtcaa catcaacggc tgcccgaact catgtgcgcg aattcaaatc

gccgacatcg gattcaaggg acagatgatc gacgacggac acggcggctc
cgtcgaaggc
ttccaggtgc atctgggcgg acacctcggc ctggatgccg
gattcggccg caaactgcgc
cagcacaagg tcaccagtga cgaactcggc
gactacatcg accgggtggt gcgcaacttc
gtcaaacacc gcagcgaagg
tgaacgcttc gcgcagtggg tcatccgggc cgaggaggac
gacctgcgat
gagcggccgc ttttttcctt

This sequence was then inserted into Geneious. The PstI and EcoRI sites were manually removed and replaced with silent mutations. A NcoI site was inserted at the start codon and an extra glycine was added after the start codon for the NcoI site to function properly. We chose glycine as it is small an unlikely to interfere with either polar or non-polar domains within the protein. We used GGC as the codon as it is the highest used glycine codon in e. coli. We also added an AvrII site on the 3’ end of the site. We then added biobrick restriction sites on the ends of the sequence (EcoRI, XbaI, SpeI, PstI). This will allow us to clone into our expression vector pACYCDuet-1 with NcoI and AvrII enzymes and into a biobrick registry with EcoRI and PstI. The insertion into pACYCDuet-1 removes both protein tags and cuts out the second MCS resulting in vector pACYCDuet-TBsirA. The resulting sequence for TB SirA follows:

catggaattc tagaccatgg gctccgcgaa ggagaacccc caaatgacca
ctgcacgtcc cgccaaggct cgaaatgagg gccagtgggc gctgggacat
cgcgagccac tcaacgccaa cgaagagctg aagaaggccg gcaacccgct
cgacgtgcgg gagcgcatcg aaaacatcta cgccaaacag ggtttcgaca
gcatcgacaa gaccgacctg cgagggcgct ttcgctggtg gggcctgtac
acccagcgtg agcagggcta cgacggcacc tggaccggtg acgacaacat
cgacaagctc gaggccaaat acttcatgat gcgggtgcgt tgcgacggcg
gcgcgctctc ggctgccgcg ctgcgcacgc tgggccagat ctcgacggag
ttcgcgcgcg ataccgccga tatctccgac cggcagaacg tgcaatacca
ctggatcgaa gtggaaaacg tccctgaaat ctggcgacgg ttagacgatg
tcggacttca gaccaccgag gcgtgcggtg actgcccgcg ggtagtgctg
ggctcgccgt tggccggcga gtcgctcgac gaagtgctcg acccgacctg
ggcgatcgag gagatcgtgc gtcgctacat cggcaagccc gacttcgccg
acttgccgcg caagtacaag accgccatct ctggccttca ggacgtcgcg
cacgagatca acgacgtcgc cttcatcggc gtcaaccatc ccgagcacgg
accaggcctg gatctgtggg tgggcggtgg actgtcgacc aacccgatgc
tggcccagcg ggtcggcgcc tgggttccac tgggcgaagt gcccgaggtg
tgggcggcgg tcacctcggt gtttcgcgac tacggctacc ggcgactgcg
cgccaaggcc cggctgaaat ttctgatcaa agactggggc atagcgaagt
tccgcgaagt gctcgaaacc gagtacctca agcgtccgct gatcgacggt
ccggcccccg aaccggtcaa gcatccgatc gaccacgtcg gggtgcaacg
actcaagaac gggctcaacg ccgtcggagt cgcccccatc gccgggcggg
tatcgggcac catcctcacg gcggtcgccg acctgatggc gcgggccggt
tccgaccgga tccggttcac cccctaccag aagctggtca tcctcgacat
tccggacgcc ttgctcgacg acttgatcgc cggtctggac gcgctggggc
ttcagtcgcg cccgtcgcat tggcgccgga acttgatggc gtgcagcggg
attgagttct gcaagttgtc attcgccgaa acccgggttc gagcacagca
tttggtgccc gagctggaac gccggcttga ggacatcaac tcgcagctcg
acgtaccgat caccgtcaac atcaacggct gcccgaactc atgtgcgcga
gttcaaatcg ccgacatcgg attcaaggga cagatgatcg acgacggaca
cggcggctcc gtcgaaggct tccaggtgca tctgggcgga cacctcggcc
tggatgccgg attcggccgc aaactgcgcc agcacaaggt caccagtgac
gaactcggcg actacatcga ccgggtggtg cgcaacttcg tcaaacaccg
cagcgaaggt gaacgcttcg cgcagtgggt catccgggcc gaggaggacg
acctgcgatg agcggccgcc taggactact agtctgcagt ttttcctt

*Mycobacterium smegmatis*

This reference also contains the cloning of sirA from T. smegmatis. We used the primers below taken from Pinto et al, blasted against the T. smegmatis genome and extracted the sirA sequence from genbank as above.

Primers:
smegsirA F      TACAGCTGATGCTCGAAGACGAGTACTTCAT

smegsirA R      CCCAAGCTTTCACGTTGCCTACCTCAAATCCGCTTCGTC

tacagctgat gctcgaagac gagtacttca tgctgcgcgt gcgctgcgat

ggtggcgcgc tgaccactgc agcgctgcgc acgctcggcg gcatctcgac

cgagttcgcg cgcgacaccg ccgacatctc cgaccgcgag aacgtccagt

accactggat ccaggtcgag aacatgcccg agatctggaa gcgcctcgac

gccgtcggcc tgcagaccac cgaggcgtgc ggcgactgcc cgcgtgtggt

cctcggctcg ccgctggccg gtgagtccct cgacgaggtg atcgacggga

cccccgcgat cgacgagatc gtgcgccgct acatcggcaa gcccgagtac

tcgaacctgc cgcgcaagtt caagaccgcg atctcggggc ttcaggacgt

ggtccacgag gtcaacgacg tcgcgttcat cggcgtcaac caccccgagc

acggcccggg cttcgacctg tgggtcggtg gcggcctgtc gaccaacccg

atgctggccc agcgcgtcgg ggtgtgggtg ccgctcgacg aggtgcccga

cgtctgggag ggcgtcgtca gcatcttccg cgactacggt taccggcgtc

tgcggtcgaa ggcgcggctg aagttcctga tcaaggactg gggcgtcgaa

aagttcaggg aagtgctgga aaccgagtac ctcaagcgcc ccctgatcga

cggcccggca cccgaaccgg tgacccgccc catcgaccac gtcggtgtgc

agaagctcaa gaacggcctc aacgccgtgg gcgtcgcccc gatcgcgggt

cgcgtctcgg gcacgatcct gaccaaggtg gccgatctcg ccgaggccgc

cgggtccgac cggatccgct tcacgccgta ccagaagctg atcatcctcg

acgtgcccga cgacaagatc gacgaactgc gcgctggcct cgacgcgctc

ggactgccgt cgacgccgtc gcactggcgc cgcaacctca tggcgtgcac

gggtatcgag ttctgcaagc tgagcttcgc cgagacccgc aagcgtgccc

aggtgctggt tcccgagctg gagaaacggc tcgacgacat caacgcccag

ctcgacgtgc ccatcacggt caacatcaac ggctgcccca actcgtgcgc

ccgtatccag gtcgccgaca tcgggttcaa gggccagatg gtcgacgatg

gcaacggccc cgaggagggt ttccaggtgc atctgggcgg cagcctgggc

ctggacagcg ggttcggccg caagctgcgc cagcacaagg tgctctcgtc

cgagctcggc gactacatcg agcgcgtcgt gcgcaacttc gtgaaacaac

gcgaggacgg cgagcgtttc gcccagtggg ccgtgcgggc cgacgaagcg

gatttgaggt aggcaacgtg aaagcttggg

As above, the sequence was imported into geneious, the biobrick restriction sites were manually removed from the sequence and restriction sites were added to the 5’ and 3’ ends in the same fashion as TB sirA. This resulted in the following sequence for synthesis:

tacagaattc tagaccatgg gcctcgaaga cgagtacttc atgctgcgcg

tgcgctgcga tggtggcgcg ctgaccacta cagcgctgcg cacgctcggc

ggcatctcga ccgagttcgc gcgcgacacc gccgacatct ccgaccgcga

gaacgtccag taccactgga tccaggtcga gaacatgccc gagatctgga

agcgcctcga cgccgtcggc ctacagacca ccgaggcgtg cggcgactgc

ccgcgtgtgg tcctcggctc gccgctggcc ggtgagtccc tcgacgaggt

gatcgacggg acccccgcga tcgacgagat cgtgcgccgc tacatcggca

agcccgagta ctcgaacctg ccgcgcaagt tcaagaccgc gatctcgggg

cttcaggacg tggtccacga ggtcaacgac gtcgcgttca tcggcgtcaa

ccaccccgag cacggcccgg gcttcgacct gtgggtcggt ggcggcctgt

cgaccaaccc gatgctggcc cagcgcgtcg gggtgtgggt gccgctcgac

gaggtgcccg acgtctggga gggcgtcgtc agcatcttcc gcgactacgg

ttaccggcgt ctgcggtcga aggcgcggct gaagttcctg atcaaggact

ggggcgtcga aaagttcagg gaagtgctgg aaaccgagta cctcaagcgc

cccctgatcg acggcccggc acccgaaccg gtgacccgcc ccatcgacca

cgtcggtgtg cagaagctca agaacggcct caacgccgtg ggcgtcgccc

cgatcgcggg tcgcgtctcg ggcacgatcc tgaccaaggt ggccgatctc

gccgaggccg ccgggtccga ccggatccgc ttcacgccgt accagaagct

gatcatcctc gacgtgcccg acgacaagat cgacgaactg cgcgctggcc

tcgacgcgct cggactgccg tcgacgccgt cgcactggcg ccgcaacctc

atggcgtgca cgggtatcga gttctgcaag ctgagcttcg ccgagacccg

caagcgtgcc caggtgctgg ttcccgagct ggagaaacgg ctcgacgaca

tcaacgccca gctcgacgtg cccatcacgg tcaacatcaa cggctgcccc

aactcgtgcg cccgtatcca ggtcgccgac atcgggttca agggccagat

ggtcgacgat ggcaacggcc ccgaggaggg tttccaggtg catctgggcg

gcagcctggg cctggacagc gggttcggcc gcaagctgcg ccagcacaag

gtgctctcgt ccgagctcgg cgactacatc gagcgcgtcg tgcgcaactt

cgtgaaacaa cgcgaggacg gcgagcgttt cgcccagtgg gccgtgcggg

ccgacgaagc ggatttgagg taggcaacgt gaaagcctag gtactagtct

gcagtggg

## Target

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Target)

### Tuesday 11th June

***Design of FdrA genes for synthesis***

**FdrA**

*Mycobacterium tuberculosis*

Reference: McLean et al. 2003. Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA. doi: 10.1042/BJ20021692

Primers:
MtbfdrA F      TATGACTCATATGCGTCCCTATTACATCGCCATCG

MtbfdrA R      ATCCGGGATCCTCAGCCGAGCCCAATCCGCAACAGC

The above primers were blasted to retrieve sequence of FprA from genebank resulting in the following sequence:

tatgactcat atgcgtccct attacatcgc catcgtgggc tccgggccgt

cggcgttctt cgccgcggca tccttgctga aggccgccga cacgaccgag

gacctcgaca tggccgtcga catgctggag atgttgccga ctccctgggg

gctggtgcgc tccggggtcg cgccggatca ccccaagatc aagtcgatca

gcaagcaatt cgaaaagacg gccgaggacc cccgcttccg cttcttcggc

aatgtggtcg tcggcgaaca cgtccagccc ggcgagctct ccgagcgcta

cgacgccgtg atctacgccg tcggcgcgca gtccgatcgc atgttgaaca

tccccggtga ggacctgccg ggcagtatcg ccgccgtcga tttcgtcggc

tggtacaacg cacatccaca cttcgagcag gtatcacccg atctgtcggg

cgcccgggcc gtagttatcg gcaatggaaa cgtcgcgcta gacgtggcac

ggattctgct caccgatccc gacgtgttgg cacgcaccga tatcgccgat

cacgctttgg aatcgctacg cccacgcggt atccaggagg tggtgatcgt

cgggcgccga ggtccgctgc aggccgcgtt caccacgttg gagttgcgcg

agctggccga cctcgacggg gttgacgtgg tgatcgatcc ggcggagctg

gacggcatta ccgacgagga cgcggccgcg gtgggcaagg tctgcaagca

gaacatcaag gtgctgcgtg gctatgcgga ccgcgaaccc cgcccgggac

accgccgcat ggtgttccgg ttcttgacct ctccgatcga gatcaagggc

aagcgcaaag tggagcggat cgtgctgggc cgcaacgagc tggtctccga

cggcagcggg cgagtggcgg ccaaggacac cggcgagcgc gaggagctgc

cagctcagct ggtcgtgcgg tcggtcggct accgcggggt gcccacgccc

gggctgccgt tcgacgacca gagcgggacc atccccaacg tcggcggccg

aatcaacggc agccccaacg aatacgtcgt cgggtggatc aagcgcgggc

cgaccggggt gatcgggacc aacaagaagg acgcccaaga caccgtcgac

accttgatca agaatcttgg caacgccaag gagggcgccg agtgcaagag

ctttccggaa gatcatgccg accaggtggc cgactggcta gcagcacgcc

agccgaagct ggtcacgtcg gcccactggc aggtgatcga cgctttcgag

cgggccgccg gcgagccgca cgggcgtccc cgggtcaagt tggccagcct

ggccgagctg ttgcggattg ggctcggctg aggatcccgg at

As previously, the sequence was imported into geneious, the biobrick restriction sites were manually removed from the sequence and restriction sites were added to the 5’ and 3’ ends in the same fashion as TB sirA. However, in the case of FdrA, the restriction sites added for cloning into the vector pCOLAduet-1 were NdeI and AvrII. As a result, there was no need to add an additional glycine after the start codon. The resulting sequence is as follows:

tatgaattct agatcatatg cgtccctatt acatcgccat cgtgggctcc

gggccgtcgg cgttcttcgc cgcggcatcc ttgctgaagg ccgccgacac

gaccgaggac ctcgacatgg ccgtcgacat gctggagatg ttgccgactc

cctgggggct ggtgcgctcc ggggtcgcgc cggatcaccc caagatcaag

tcgatcagca agcaattcga aaagacggcc gaggaccccc gcttccgctt

cttcggcaat gtggtcgtcg gcgaacacgt ccagcccggc gagctctccg

agcgctacga cgccgtgatc tacgccgtcg gcgcgcagtc cgatcgcatg

ttgaacatcc ccggtgagga cctgccgggc agtatcgccg ccgtcgattt

cgtcggctgg tacaacgcac atccacactt cgagcaggta tcacccgatc

tgtcgggcgc ccgggccgta gttatcggca atggaaacgt cgcgctagac

gtggcacgga ttctgctcac cgatcccgac gtgttggcac gcaccgatat

cgccgatcac gctttggaat cgctacgccc acgcggtatc caggaggtgg

tgatcgtcgg gcgccgaggt ccgcttcagg ccgcgttcac cacgttggag

ttgcgcgagc tggccgacct cgacggggtt gacgtggtga tcgatccggc

ggagctggac ggcattaccg acgaggacgc ggccgcggtg ggcaaggtct

gcaagcagaa catcaaggtg ctgcgtggct atgcggaccg cgaaccccgc

ccgggacacc gccgcatggt gttccggttc ttgacctctc cgatcgagat

caagggcaag cgcaaagtgg agcggatcgt gctgggccgc aacgagctgg

tctccgacgg cagcgggcga gtggcggcca aggacaccgg cgagcgcgag

gagctgccag ctcagctggt cgtgcggtcg gtcggctacc gcggggtgcc

cacgcccggg ctgccgttcg acgaccagag cgggaccatc cccaacgtcg

gcggccgaat caacggcagc cccaacgaat acgtcgtcgg gtggatcaag

cgcgggccga ccggggtgat cgggaccaac aagaaggacg cccaagacac

cgtcgacacc ttgatcaaga atcttggcaa cgccaaggag ggcgccgagt

gcaagagctt tccggaagat catgccgacc aggtggccga ctggctagca

gcacgccagc cgaagctggt cacgtcggcc cactggcagg tgatcgacgc

tttcgagcgg gccgccggcg agccgcacgg gcgtccccgg gtcaagttgg

ccagcctggc cgagctgttg cggattgggc tcggctgagg atccctagga

ctagtctgca gttacg

*Mycobacterium smegmatis*

We didn't find a reference of M. Smegmatis FprA being cloned into E. coli so we used the following sequenced data:

atgcgcccgt accacgtagc gatcgtcggc tcaggaccct ccggattctt

tgctgccgca tcgctgctga agtttgccga ctctcaaccc gaccgcgatg

tccgcgtgga catgctcgag atgctgccga ccccgtgggg tctggtgcgc

tccggcgtcg ctcccgacca tccgaagatc aagtcgatca gcgcccagtt

cgagaagacc gcggccgacc cccgcttccg gttcttcggc aacgtccggg

tcggtgagca cgtccaaccg ggcgaactcg ccgaacgcta cgacgccgtc

gtctacgcca ccggcgcaca gtccgaccgc gcgctgaaca tccccggcga

ggagctgccg ggcagcgtcg cggccgtcga cttcgtgggt tggtacaacg

cgcatccgca tttccgcgag atggcgcccg atctctccgg cgggcgcgcc

gtggtggtcg gcaacggcaa cgtcgcactc gatgtcgcac gcatcctggt

cagcgatccg aaggcgttgg ccaacaccga cattgccgac catgcgctgg

acaagctgga cacgcgcggt gtggacgagg tcgtggtgct cggccggcgt

ggtccgctgc aggcgacgtt caccacgctg gagttgcgtg aactcggcga

catggagggc ctcggcgacg tcgacgtgat cctggatccg gccgatttcg

ccgacatcac cgacgaggat ctcgaagccg cgggcaagac cgtcaagcag

aacatcaagg tgctgcgcgg ttacgccgag cgggaaccgc gaggcgccaa

gcggcgcatc gtgttccggt tcttcacctc accgatcgag ctgcgcggcg

aggaccgcgt ggaatcgatc gtgttgggac gcaacgaact cgtgcgcggc

gccgacggca ggatggtggc caaggacacc ggtgagcgcg aagaactgcc

ggcacagctg gtggtgcgtg cggtcggata ccgcggcgtg ccgactccag

gcctgccgtt cgacgagcgt tcgggcacca tcccgcacac cgacggccgc

gtcgagggca gcgccaacga gtacgtggtg ggctggatca agcgcggacc

gtccggcgtc atcggcagca acaagaagga ctcacaggac accgtcaaca

ccctgctcga cgacctcgcc gcggcccggc tgccccagcg cgggcccgac

cactccgaga agctcgcggc gtggttgctg gaacgtcagc ccaaggtggt

cacgggcgag cactggaagc tgatcgacga ctacgagcgc gccgcgggcg

aaccgaccgg ccgaccccgg gtgaagctgg cgagcgtggc tgaactgctg

cgcgtgggcc acggctga

As above, the sequence was imported into geneious, the biobrick restriction sites were manually removed from the sequence and restriction sites were added to the 5’ and 3’ ends in the same fashion as TB sirA. Again, the restriction sites added for cloning into the vector pCOLAduet-1 were NdeI and AvrII. As a result, there was no need to add an additional glycine after the start codon. The resulting sequence is as follows:

tatgaattct agatcatatg cgcccgtacc acgtagcgat cgtcggctca

ggaccctccg gattctttgc tgccgcatcg ctgctgaagt ttgccgactc

tcaacccgac cgcgatgtcc gcgtggacat gctcgagatg ctgccgaccc

cgtggggtct ggtgcgctcc ggcgtcgctc ccgaccatcc gaagatcaag

tcgatcagcg cccagttcga gaagaccgcg gccgaccccc gcttccggtt

cttcggcaac gtccgggtcg gtgagcacgt ccaaccgggc gaactcgccg

aacgctacga cgccgtcgtc tacgccaccg gcgcacagtc cgaccgcgcg

ctgaacatcc ccggcgagga gctgccgggc agcgtcgcgg ccgtcgactt

cgtgggttgg tacaacgcgc atccgcattt ccgcgagatg gcgcccgatc

tctccggcgg gcgcgccgtg gtggtcggca acggcaacgt cgcactcgat

gtcgcacgca tcctggtcag cgatccgaag gcgttggcca acaccgacat

tgccgaccat gcgctggaca agctggacac gcgcggtgtg gacgaggtcg

tggtgctcgg ccggcgtggt ccgcttcagg cgacgttcac cacgctggag

ttgcgtgaac tcggcgacat ggagggcctc ggcgacgtcg acgtgatcct

ggatccggcc gatttcgccg acatcaccga cgaggatctc gaagccgcgg

gcaagaccgt caagcagaac atcaaggtgc tgcgcggtta cgccgagcgg

gaaccgcgag gcgccaagcg gcgcatcgtg ttccggttct tcacctcacc

gatcgagctg cgcggcgagg accgcgtgga atcgatcgtg ttgggacgca

acgaactcgt gcgcggcgcc gacggcagga tggtggccaa ggacaccggt

gagcgcgaag aactgccggc acagctggtg gtgcgtgcgg tcggataccg

cggcgtgccg actccaggcc tgccgttcga cgagcgttcg ggcaccatcc

cgcacaccga cggccgcgtc gagggcagcg ccaacgagta cgtggtgggc

tggatcaagc gcggaccgtc cggcgtcatc ggcagcaaca agaaggactc

acaggacacc gtcaacaccc tgctcgacga cctcgccgcg gcccggctgc

cccagcgcgg gcccgaccac tccgagaagc tcgcggcgtg gttgctggaa

cgtcagccca aggtggtcac gggcgagcac tggaagctga tcgacgacta

cgagcgcgcc gcgggcgaac cgaccggccg accccgggtg aagctggcga

gcgtggctga actgctgcgc gtgggccacg gctgacctag gactagtctg

cagttacg

## Target

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Target)

### Wednesday 12th June

***Design of FdxC and FdxA genes for synthesis***

**FdxC**

*Mycobacterium tuberculosis*

Reference: Cole, S.T. et al. 1998. Deciphering the biology of Mycobacterium tuber-
culosis from the complete genome sequence. doi:10.1038/31159.

We couldn’t find a reference of FdxC being cloned into E. coli and so we took the sequence data from genbank which is as follows:

gtgacgtaca cgatcgccga accctgtgtc gacatcaagg acaaggcatg

cattgaggag tgcccggtcg attgcatcta cgagggcgcc cggatgctgt

atatccaccc cgacgaatgc gtcgactgtg gggcttgcga gccggtctgc

cccgttgaag ctatcttcta cgaagacgat gtgcccgaac agtggagcca

ttacacccag atcaacgccg atttcttcgc cgagctggga tcgccgggcg

gtgcggccaa ggttggcatg accgagaacg acccgcaagc ggtcaaggat

ctggcgccgc agagcgagga cgcctga

The Gtg was replaced with the NcoI cut site and a glycine codon (ccatgggc) and a HindIII site was added to the end of the sequence. The biobrick restriction sites were then added to the ends of the sequence resulting in the following sequence:

cttagaattc tagaccatgg gcacgtacac gatcgccgaa ccctgtgtcg

acatcaagga caaggcatgc attgaggagt gcccggtcga ttgcatctac

gagggcgccc ggatgctgta tatccacccc gacgaatgcg tcgactgtgg

ggcttgcgag ccggtctgcc ccgttgaagc tatcttctac gaagacgatg

tgcccgaaca gtggagccat tacacccaga tcaacgccga tttcttcgcc

gagctgggat cgccgggcgg tgcggccaag gttggcatga ccgagaacga

cccgcaagcg gtcaaggatc tggcgccgca gagcgaggac gcctgaagct

tgactagtct gcagaaatg

**FdxA**

*Mycobacterium smegmatis*

Reference: Ricagno S. et al. 2008. The crystal structure of FdxA, a 7Fe ferredoxin from Mycobacterium smegmatis. http://dx.doi.org/10.1016/j.bbrc.2007.06.013
http://www.metacyc.org/MSME246196/NEW-IMAGE?type=GENE-IN-MAP&object=GJ4Y-1124

Sequence:

atgacatacg tgatcggccg gccgtgcgtg gacgtcaaag accgcgcatg

cgtggatgag tgcccggtcg actgcatcta cgagggcgcg cggatgctct

acatccaccc cgacgaatgt gtcgactgtg gcgcgtgtga acccgtgtgc

ccggtggagg cgatctacta cgaggacgac ctgcccgagg atctgcagcc

gtaccaggag gagaacgcga agttcttcac cgatgtcctg cccgggcgtg

cccaaccgct cggatcaccg ggcggtgccg cgaaactcgg tgtcgtggac

gccgatacgc ccatggttgc ggagctaccg ccgcaggggg attga

The NcoI cut site and a glycine codon (ccatgggc) replaced the atg in green and a HindIII site was added to the end of the sequence. The biobrick restriction sites were then added to the ends of the sequence resulting in the following sequence:

cttagaattc tagaccatgg gcacatacgt gatcggccgg ccgtgcgtgg

acgtcaaaga ccgcgcatgc gtggatgagt gcccggtcga ctgcatctac

gagggcgcgc ggatgctcta catccacccc gacgaatgtg tcgactgtgg

cgcgtgtgaa cccgtgtgcc cggtggaggc gatctactac gaggacgacc

tgcccgagga tcttcagccg taccaggagg agaacgcgaa gttcttcacc

gatgtcctgc ccgggcgtgc ccaaccgctc ggatcaccgg gcggtgccgc

gaaactcggt gtcgtggacg ccgatacgcc catggttgcg gagctaccgc

cgcaggggga ttgagcctag gtactagtct gcagtggg

## Target

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Target)

### Tuesday 18th June

***In Silico Cloning***

We used geneious to perform in silico cloning on our constructed gene sequences to ensure that they were properly made. SirA and pACYCDuet-1 were both cut with NcoI and AvrII and then the large fragments were extracted and ligated together to form vector pD001. FdxA and pETDuet-1 were both cut with NcoI and HindIII and the large fragments were extracted and ligated together to form vector pD002. FprA and pD002 were both cut with NdeI and AvrII and the large fragments were extracted and ligated together to form vector pD003. Please see Geneious files on Google Drive (or send request for files) for exact sequences.

## Target

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Target)

### 24th June

***Codon Optimization***

The ORF of M. Smegmatis SirA, FprA, and FdxA were codon optimized for E. coli K-12 using JCat (www.jcat.de) with options to avoid using rho-independent terminators, prokaryotic RBS, and restriction sites used in vector construction (AvrII, HindIII, NdeI, NcoI) and Biobrick restriction sites (EcoRI, PstI, SpeI, XbaI). The optimization charts are shown below:

## Target

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Target)

### Monday 1st July

***Media was made, Strains sD001-sD004 were received, inoculated, and put into stock and catalog.***

4 strains were received from Jake:

* E. coli: BL21 (DE3) ko20 ΔcysI, Δfpr, ΔydbK* E. coli: NEBTurbo zmSIR Chloramphenicol* E. coli: NEBTurbo zmFNR Spectinomycin* E. coli: NEBTurbo soFD, zmSIR Chloramphenicol

##### Media and Glycerol stock were prepared

###### Media Preparation

3 500ml bottles of LB broth and LB agar were prepared by standard methods.
 12.5g/500ml powder/water for broth and 20g/500ml powder/water for agar.
Bottles were autoclaved. Additionally 1 flask of 500ml of LB broth was made in the same fashion.

###### Glycerol Stocks

Single colonies from agar plates were picked and used to innoculate 5ml LB broth overnight.
 750ml of overnight culture was added to 250ml of 60% glycerol in a cryotube.
 Two sets of Glycerol stocks were used for each of sD001, sD002, sD003, sD004; one set was frozen at -20ºC and the other set was frozen at -80ºC.

## Target

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Target)

### Tuesday2nd July

***MiniPrep: Plasmids pD004, pD005 and pD006 were extracted from NEBturbo vectors***

Plasmid Extraction
Plasmids pD004 (pCDF.ew12 zmFNR Spectinomycin), pD005 (pACYC.ew13 soFD, zmSIR Chloramphenicol), pD006 (pACYC.ew17 zmSIR Chloramphenicol) were extracted from NEBTurbo cells using a Thermo Scientific GeneJet Plasmid mini prep kit as described in protocol (). Lacking Resuspention solution we used some from a different mini prep upstairs. Plasmids were eluted in 100ul of nanopure water and frozen at -20ºC.
More notes here tra la lalal

## Target

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Target)

### Wednesday 3rd July

***Chemically Competent Cells : BL21 (DE3) dCysI dFpr dydbk, NEBturbo stocks***

Chemically Competent BL21 (DE3) Cells were made
We grew 5ml of sD001 cells overnight from a single colony. This 5ml was used to innoculate 500ml of LB broth.
 Broth was incubated for 1.5h and optical density was read at 0.62 OD600.
Cells were alloquoted into 10 falcon tubes (50ml each) and centrifuged at 4000xg for 20 minutes at 4C.
 Supernatant was removed and cells were resuspended in 200ml of Buffer 1 (4x 50ml) and left on ice for 20 minutes.
 Cells were centrifuged again for 20 minutes at 4000xg.
 Supernatant was removed and cells were resuspended in 12ml Buffer 2.
 Cells were alloquoted at 500ul into microcentifuge tubes and frozen at -80C.

Buffer 1: 50mM CaCl2

Buffer 2:

* 0.53ml 2M CaCl2
  * 2.8ml 60% Glycerol
    * 8.67ml sterile H2O

This is the correct Chemical Competent Cells protocol (Similar to Protocol 2) , that we should have used instead of the one above:

1. Take a single colony of E. coli cells and inoculate 5ml of LB broth.
    Incubate over night at 37C and 200rpm and innoculate about 500ml to 1L sterile LB broth.
    DO NOT ADD antibiotics since these cells do not have a plasmid in them.
    Work as sterile as possible.- Inoculate about 300-400ml sterile LB broth with 500ul of preculture.- Grow the cells on a shaker at 37C until they reach an OD @ 600nm of 0.3 to 0.4 (1cm pathlength of the cuvette).- Centrifuge at 3000xg for 10 minutes at 4C. Ice down 100mM CaCl2 and 100mM MgCl2 solutions at this point.- Gently resuspend the bacteria pellet on ice in 50ml of ice cold 100mM MgCl2, taking 3-5 minutes for this procedure.
            Centrifuge the cell suspension at 3000xg for 10 minutes at 4C.- Resuspend the bacteria pellet on ice 50ml of ice cold 100mM CaCl2, incubate the cells for 10 minutes on ice.- Centrifuge the cell suspension at 3000xg for 10 minutes at 4C and resuspend the cell pellet in 4ml of ice cold, sterile 100mM CaCl2 in 15%(w/v) glycerol.
               Dispense in 50uL aliquots and freeze cells at -80C.

Chemically competent cells (NEB Turbo)

Prepared via iGEM chemically competent protocol with following changes.
 500ul of preculture used to inoculate 500ml LB broth. Grown at 37C for 3h hours to an OD600 of 0.536.
Cells centrifuged at 3200 rpm on old centrifuge for 10m at 4C. Pellets resuspended in 80ml of CCMB80 buffer.
 Incubated on ice for 20m. Centrifuged for 10m at 3200 rpm for 10m at 4C.
Pellets resuspended in 5ml of CCMB80 buffer. Aliquoted into Microcentrifuge tubes and frozen at -80C.

## Target

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Target)

### Thursday 4th July

***Heat Shock Transformation***

**Preparation of Agar Plates**
LB Agar was melted; 100ml was used to make 8 ampicillin plates,
100ml was used to make 8 spectinomycin plates and the remaining was used to make 20 chloramphenicol plates.
Antibiotic concentrations are: Ampicillin 50ug/ml, Spectinomycin 50ug/ml, Chloramphenicol 34ug/ml.

**Heat Shock Transformation**
sD001: BL21 (DE3) with deletions, was transformed with the following plasmids and plasmid combinations:

* sD001+pD004 ( FNR, Spectinomycin)* sD001+pD005 ( SIR, Fdx, Chloramphenicol)* sD001+pD006 (Sir, Chloramphenicol)

Transformation was done according to protocol 1 with the following specifications:
BL21 (DE3) chemically competent cells were thawed on Ice. 1ul of plasmid DNA was added to a tube of BL21 (DE3) cells.
Cells were incubated on ice for 30 minutes. Cells were transfered to heating block at 42C for 45s.
 Cells were returned to ice for 2 minutes. 1ml of LB Broth was added to the cells and they were incubated at 37C for 1h.
Cells were then plated on agar with appropriate antibiotics (specified previously).
These transformations worked; single colonies were lifted from plates, entered to catalog and stocked in glycerol stock (protocol 3).

Catalog names:

* sD006= sD001+pD004 ( FNR, Spectinomycin)* sD007= sD001+pD005 ( SIR, Fdx, Chloramphenicol)* sD008= sD001+pD006 (Sir, Chloramphenicol)

## Target

 [ASDF](https://2013.igem.org/Team%3AParis_Bettencourt/Project/Target)

### Friday 5th July

***Planning transformations for the next week***

We
decided to partly reproduce Figure 3C from:
<http://www.jbioleng.org/content/5/1/7>:

![](https://lh5.googleusercontent.com/EKwdYjSSLfO_91W7fgqGLKIPkkGjAMWWrfB0GcvR5o1wlQJsBaa5cqajDhreNfAi5ZS1cLaMgKrws8sn6VhEGAAgIfEajdqALZCCNgrDG9NSD0AoEV_kLXdO)

So,
we would pursue to grow the following strains on a minimal media M9
plate, supplemented with all amino acids

aside
from the sulfur containing ones, and sulfite ans the only sulfur
source:

We
already thought we have all of the strains we need, however, an
overnight culture was launched of a few single colonies from this
double plasmid strain (sD001+pD004+pD005, which supposedly had all 3
genes SIR FNR and Fdx) and it didn't grow.

This
most likely occurred because the strains didn't have *Spec*antibiotics
resistance, as the other spec resistant strain containing

the
FNR gene (pD004), was found not to be resistant as well.

![](https://static.igem.org/mediawiki/2013/thumb/9/91/Wishplate.jpg/800px-Wishplate.jpg)
*Illustration
1: Strains will be grown on a minimal media M9 plate, supplemented
with all amino acids aside from the sulfur containing ones; plates
will have increased glucose, and sulfite as the only sulfur source.
Growth is expected only for WT and the strain containing all three
alternative genes of sulfur reduction.*

Therefore,
we would  re-make:

* **sD001+pD004+pD005**
  E.coli:ΔcysI,
  Δfpr, ΔydbK::
  **soFD**,
  **zmSIR**;Chloramphenicol
  ,**zmFNR**;
  Spectinomycin- with
  all three genes.

  * **sD001+pD004:**E.coli:ΔcysI,
    Δfpr, ΔydbK::
    **zmFNR**;
    Spectinomycin

and
make

* **sD001+pD004+pD006**
  E.coli:
  ΔcysI,
  Δfpr, ΔydbK::**zmFNR**;
  Spectinomycin, **zmSIR**;
  Chloramphenicol

These
transformations will be done by using two strains of competent cells
prepared using Protocol 2 :

* sD001
  competent cells were transformed with:

  + pD004

    + Double
      transformation: pD005 +pD004* sD007
    (sD001+pD005) competent cells were transformed with:

    + pD004

These
transformations will be done by heat shock using protocol 1.