iGEM 2011 — Team Colombia

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# Team:Colombia/Notebook

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| Contents   * [1 Colombia @ iGem Notebook](#Colombia_.40_iGem_Notebook)   + [1.1 June](#June)     - [1.1.1 June 30:](#June_30:)   + [1.2 July](#July)     - [1.2.1 July 1](#July_1)     - [1.2.2 July 5](#July_5)     - [1.2.3 July 6](#July_6)     - [1.2.4 July 7](#July_7)     - [1.2.5 July 8](#July_8)     - [1.2.6 July 11](#July_11)     - [1.2.7 July 12](#July_12)     - [1.2.8 July 14](#July_14)     - [1.2.9 July 15](#July_15)     - [1.2.10 July 19](#July_19)     - [1.2.11 July 21](#July_21)     - [1.2.12 July 22](#July_22)     - [1.2.13 July 27](#July_27)     - [1.2.14 July 30](#July_30)   + [1.3 August](#August)     - [1.3.1 August 3](#August_3)     - [1.3.2 August 5](#August_5)     - [1.3.3 August 6](#August_6)     - [1.3.4 August 9](#August_9)     - [1.3.5 August 11](#August_11)     - [1.3.6 August 16](#August_16)     - [1.3.7 August 17](#August_17)     - [1.3.8 August 25](#August_25)     - [1.3.9 August 29](#August_29)     - [1.3.10 August 30](#August_30)     - [1.3.11 August 31](#August_31)     - [1.3.12 September 1](#September_1)     - [1.3.13 September 3rd](#September_3rd)     - [1.3.14 September 12th](#September_12th)     - [1.3.15 September 13th](#September_13th)     - [1.3.16 September 14th](#September_14th)     - [1.3.17 September 15th](#September_15th) |

# **Colombia @ iGem Notebook**

Here you can find our daily work in the Lab!

## **June**

### June 30:

* Biobricks 1, 2, 3, 4 and 5 were resuspended
* Miniprep Solutions (I, II and III) were prepared.

## **July**

### July 1

* Biobricks 1, 2, 3, 4 and 5 were resuspended.

### July 5

* Biobrick 2 presented no colonies.
* Colonies from bricks 1, 3, 4 and 5 were stinged.

> :   *Task:*
> :   To make LB medium (15x25mL)

### July 6

* Biobricks 1, 3 and 4 were twice plated.
* Brick 5 didn't grow up.
* We've added Ampiciline (3.75mL) and Kanamicine (1.875mL) to the boxed from the previous day.
* LB medium was prepared (15x25mL).
* Liquid LB was prepared (400mL).

> :   *Task:*
> :   To add ampiciline and tetracicline to the boxes.
> :   Claim the liquid LB
> :   Scrape ....
> :   Sting biobricks 2 and 5 (no clons)
> :   Pick up the tubes with Merceditas

### July 7

* Clons 2 and 5 didn't work out.
* Clons 1, 3 and 4 were planted in LB liquid.
* E. Coli inoculation in Coffee
* Kanamicine resistence plasmid: 0.2 optic density.
* Direct inoculation x 2 and C(-) MgCl2.

> :   *Task:*
> :   Print electroporation protocol.
> :   Ask Juan D. Olarte about the inoculation in coffee.
> :   Minipreps for confirmation of 1, 3 and 4.
> :   Competent cells for clons 2 and 5.

### July 8

* Minipreps for 1, 3 and 4 were made.
* Plant sheets of coffee into different plates: Each sheet was cut in the middle and they were incubated at 25°C and 37°C LB+Kan.

> :   *Task:*
> :   To finish the minipreps from the addition of RNAsa.
> :   Check the E. Coli growth in coffee.

### July 11

* Minipreps have been finished. They've been planted in gel: Low concentration (25 ng/uL).
* Transformation protocol in chemical cells.

> :   *Task:*
> :   Prepare 250 mL of SOC medium
> :   Print the Transformation protocol for chemical cells.

### July 12

* Strains 1 and 4 have been conserved.
* LB liquid culture was prepared again for brick 3.
* E. Coli growth results.

> :   *Task:*
> :   Print the Transformation protocol for chemical cells.
> :   Competent cells for clons 2 and 5.
> :   Preserve brick 3.
> :   Confirm bricks 1, 3 and 4 (Digestion)
> :   Resuspend all Biobricks.
> :   Check the E. Coli growth in coffee.

### July 14

* E. Coli didn't grow up on the sheets.
* Chemical competent cells were made (DH5α).
* Brick 3 was left to grow in liquid medium.
* Bricks 2, 5, 6,7, 8, 9 and 10 were transformed and plated.

> :   *Task:*
> :   Print the Transformation protocol for chemical cells.
> :   Preserve brick 3.
> :   Confirm all biobricks (Digestion)
> :   Sting the transformed bricks.
> :   Add antibiotic to the mediums made today.

### July 15

* Brick 3 was preserved in Revco.
* Bricks 2, 5 and 8 were stinged.
* 25 LB+Kan boxes x 25 mL.
* No colonies in brick 6.
* Bricks 7, 9 and 10 were contaminated.

> :   *Task:*
> :   Print the chemical cells protocol.
> :   Confirm all biobricks (Digestion).

### July 19

* Pass strain of Vibrio fischeri to blood agar base.
* Check the growth of the isolates
* Pass the transformed bricks 6, 7, 9 and 10.
* Pass the isolates the solid media to liquid media (2,4,5 and 8)

### July 21

* Digestion to confirm No. 1, 3 and 4

|  |  |  |
| --- | --- | --- |
| Reactives | 1X | 6X |
| H2O | 29,4µL | 160,4 µL |
| Buffer N. 3 | 4 µL | 24 µL |
| EcoRI | 0,3 µL | 1,8 µL |
| PstI | 0,3 µL | 1,8 µL |
| DNA | 7 µL | 40 µL |

### July 22

* Minipreps
* Electrophoresis of the digestions

> :   *Task:*
> :   Confirm minipreps
> :   Re-suspend primers

### July 27

* To prepare LB and SOC medium and autoclaved
* The bricks 7, 9 and 10 were again transformed and plated
* Plate the brick 6.

### July 30

* Minipreps with RNase.
* Agarose gel Electrophoresis—Results were not obtained. **REPEAT!!**

---

## **August**

### August 3

* PCR 16S to DNA Vibrio fischeri U. Nacional
* To expected a band of 1400 bp.

|  |  |  |
| --- | --- | --- |
| Reactives | 1X | 3X |
| H2O | 6,8µL | 20,4µL |
| Bµffer | 1µL | 3µL |
| MgCl2 | 0,8µL | 2,4µL |
| dNTPs | 0,2µL | 0,6µL |
| Fw7 | 0,2µL | 0,6µL |
| Rv49 | 0,2µL | 0,6µL |
| Taq | 0,1µL | 0,3µL |
| DNA | 1µL | - |
|  | 10µL |  |

**PCR Conditions**

|  |
| --- |
| 94 C for 5 min |
| 95 C for 50 sec |
| 55 C for 45 sec 35X |
| 72 C for 1:30 min |
| 72 C for 12 min |
| 12 C forever |

### August 5

* Re-suspend primers

100µM → 10 µM

Example:
igem 1 → 36.1 nm → 361 µL H2O
igem 2 → 32.2 nm → 322 µL H2O ➱ Vortex

Vf= 50 µL
Ci= 100 µL
Cf= 10 µL
Vi= ?

Vi= 5 µL

* Diluted DNA Vibrio fischeri

1244.1 ng/ µL

Ci= 1244.1 ng/ µL

Cf= 25 ng/ µL

Vf= 50 µL

Vi= 1 µL + 49 µL H2O

### August 6

* Solutions 2 and 3 of miniprep
* Transformations of 2, 5, 7 and 13
* Check primers

**Inventory**

The petri dishes with bacteria are in Q401

**PCR genes (sensor, CBP and chitoporin) of Vibrio fischeri with Pfx**

|  |  |  |  |
| --- | --- | --- | --- |
| Name | Dir | Gene | TM |
| Igem 1 | Fw | sensor | 48,8 C |
| Igem 2 | Rv | sensor | 50,8 C |
| Igem 3 | Fw | CBP | 48,9 C |
| Igem 4 | Rv | CBP | 49,1 C |
| Igem 5 | Fw | Chitopor | 49,3 C |
| Igem 6 | Rv | Chitopor | 49,4 C |

### August 9

* Minipreps:

|  |  |
| --- | --- |
| 1 | ✔ |
| 2 | ✕ |
| 3 | so-so |
| 4 | ✔ |
| 5 | ✕ |
| 8 | so-so |
| 9 | so-so |
| 10 | so-so |

### August 11

* PCR chiA of Vibrio fischeri with Pfx

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Name | Dir | Gene | Tm | Lenght |
| Igem 7 | Fw | ChiA | 55,4 C | 3378 pb |
| Igem 8 | Rv | ChiA | 53,3 C | 3378 pb |

**PCR Conditions**

|  |  |
| --- | --- |
| Denaturation Step | 94 C for 3 min |
| Denaturation | 95 C for 45 sec |
| Annealing | 53,5 C for 45 sec 35X |
| Extension | 68 C for 2:10 min |
| Final Elongation | 68 C for 6 min |
|  | 12 C forever |

### August 16

* Minipreps bricks 5 and 10
* Plate the bricks 8 and receptor plasmid ( purple colonies)

> *Task:*
>
> :   Electrophoresis minipreps bricks 5 and 10
> :   Growth in liquid media LB colonies of the bricks 8 and receptor plasmid -- minipreps
> :   PCR of Vibrio fischeri: sensor, chitoporin and ChiA
> :   Digestions of the bricks

### August 17

* Brick 8 didn’t grow
* We made Chloramphenicol stock solution.

### August 25

**Plasmid 1**

**BACKBONE pSB1C3**
1. Cut with NotI and SpeI backbone
2. Phosphate backbone

**SENSOR**
Cut with:
1. Amplification Senor and Adelinate
2. Cloning into pGEM Teasy
3. Cut with NotI and XmaJI Sensor
4. Insert into backbone= backbone 1 (use ligase T4)

Terminator:
1. Miniprep terminator (21)
2. Cut with PstI and SpeI
3. Cut backbone 1 with XbaI
4. Insert terminator into backbone 1 = backbone 2 (use ligase T4)

CBP:
(XhoI-FW Sensor)-(XmaJI RVSensor) –(XbaI Terminador-PstI)- (XbaI FW CBP)-(NheI RV CBP) – (XbaI RV Chitoporin)-(PstI FW Chitoporin)

**Plasmid 2**

7-18-(11/12/19)- 8- (11/12/19)-6 - 21- 1- 18-(11/12/19)-14-(11/12/19)-9-21

**Plasmid 3**

2-(11/12/19)6-T-1-(11/12/19)-5 -T-1-4-T

**7:30 pm**

* *Vibrio fischeri* PCR

* Attempt to achieve the PCR amplification of the ChiA and Chitoporin genes.

* Amplification results are to be visualized on a gel tomorrow.

* The amplification of ChiA and Chitoporin genes didn´t work. : ( REPEAT!!!

* Digestions for Brick 2 and 8A employed the EcoRI and BSA buffer.

|  |  |  |
| --- | --- | --- |
| Reactives | 1X | 2X |
| H2O | 32,2 | 64,4 |
| Buffer 10x | 4uL | 8uL |
| ADN | 3uL | 3uL |
| EcoRI | 0.4uL | 0.8uL |
| BcuI(SpeI) | 0.4uL | 0.8uL |

* Digestions for 3 hours at 37C and inactivated at 65C for 15 seconds.

**Brick 8** ok (750 bp)

**Brick 2**  : (

### August 29

PCR of chitinase. :(
Vibrio fischeri ES114 grown at 25 C

### August 30

PCR reactions of Chitoporin (chiP) and chitinase (chiA) using temperature gradient were made today. Two The DNA comes from picked colonies of Vibrio fischeri ES114 (Grown 25 C) and the following reactives were used:

|  |  |  |
| --- | --- | --- |
| Reactives | 1X | 3X |
| Taq | 0.1µL | 0.3µL |
| MgCl2 | 0.8µL | 2.4µL |
| dNTPs | 0.44µL | 1.32µL |
| Fw | 0.44µL | 1.32µL |
| Rv | 0.44µL | 1.32µL |
| DMSO | 1µL | 3µL |
| Taq | 0.1µL | 0.3µL |

### August 31

* Plate the bricks 8, terminator and GFP and make it grow in liquid media (LB)
* PCR Doesn`t work out !

*To do list*
Pick up of liquid media and grow with antibiotic 5 colonies for each plate (8, terminator and GFP) 1:00 pm

- To make a list of the enzymes digested for each brick PLEASE!!!!!!!!!!!!!
- The strong RBS is ….BBa\_B0030
- The moderate RBS is BBa\_B0034
- The weak RBS is BBa\_B0032

Tomorrow
- Miniprep of the bricks 8, terminator and GFP
- Extraction DNA Vibrio fischeri ES114
- Digestions of bricks

### September 1

2
pCAT

Test the brick No. 4 for hypersensitive response in plants.

### September 3rd

Vibrio fischeri DNA extraction with PureLink™ Genomic DNA Mini Kit

### September 12th

Ligation Sensor into pGEM Teasy

### September 13th

* Insert of Brick No. 6 extracted from gel. No other results.

* Transformation Sensor PgemTeasy

### September 14th

4 colonies of transformation into PGEM-Teasy

### September 15th

Miniprep colonies PGEMTeasy

NotI fermentas Buffer O
XbaI promega Buffer D
EcoRI Promega Buffer H
PstI promega Buffer H
SpeI Fermentas Buffer Tango

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