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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

Team:Colombia/Notebook