Team:Wageningen UR/Notebook/Proj1/July

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Building a Synchronized Oscillatory System

July - Synchronized Oscillatory System

July 5

We created and isolated the following parts: C0061-B0015 & F2621-E0422 & F2621-I0460 & R0062-I0460.

July 6

Digestions and ligations :)

July 8

Colony PCR from the ligations made on July the 6th.

July 11

More digestions and subsequent ligations.

We also created and isolated the following parts: K182102-C0061-B0015 & R0062-I0460.

July 13

Colony PCR from the ligations made on July the 11th. BioBrick parts were digested using different combinations of restriction enzymes and run on a gel.

July 14

Ligations were performed using the digestions from the 13th.

July 15

Colony pcr to verify the ligations of the 14th and subsequently liquid cultures of the positive colonies were made. We also created and isolated the following parts: K318511-S01003 & K182102-C0061-B0015-R0062-I0460 & R0040-K082014 & F2621-K082014-F2621 & F2621-E0422-F2621-K082014 & R0040-E0422 & F2621-K082014-F2621-E0422 & E0422-F2621-K082014 & & F2621-E0422-F2621-I0460 & R0040-K082014

July 20

Digestions, ligations, transformations.

July 22

PCR screen of the ligations from July 20th.

July 23

Digestions and ligations.

July 25

Transformations of the ligations from the 23rd.

July 26

We created and isolated the following parts: K182102-c0061-B0015 & R0062-I0460 & F2621-K082014 & F2621-I0460. More digestions, ligations and transformations.

July 27

Colony pcr to verify yesterday’s ligations.

July 28

Digestions, ligations, transformations.

July 29

Colony PCR and making liquid cultures

Also making new digestions and ligations.

July 30

Isolating the parts from the liquid cultures made on the 29th. Besides that, we also inoculated 500 mL erlenmeyers with 100 ml LB + 100 ug/ul. E. coli was grown overnight on LB + amp plates and directly used as inoculum. All cultures were done in duplo. E. coli strains used were: ptet-GFP R0062-GFP F2621-GFP RBS-GFP

Cultures were put in a shake incubator at 30°C, 300 rpm. Temperature was set to 30 degrees as GFP is degraded at higher temperatures. The OD600s of every culture were measured 2 hours after inoculation and subsequently each hour, by taking 1 mL samples from the culture. the OD was measured with a Hitachi U1500 spectrophotometer. LB+amp was taken as blank. Fluorescence was measured in a 96-wells plate on a fluorescent plate reader (Molecular Devices, M2 (toxicology). First blank measurements were done with 96 wells plates containing 50 ul LB+amp. The output corresponding with the specific wells was saved and used for further calculations.

OD600 data presented below

Sample	no. 1 (2 hours)	no. 2 (2 hours)	no. 1 (4 hours)	no. 2 (4 hours)
RBSGFP	min 0.001	min 0.001	0.021	0.004
R0062	min 0.016	min 0.011	0.001	0.020
ptetGFP	min 0.006	min 0.004	0.011	min 0.003
F2621GFP	min 0.014	min 0.003	0.009	0.011

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+[[File:Jul.png]]
+'''July 5'''
+We created and isolated the following parts: C0061-B0015 & F2621-E0422 & F2621-I0460 & R0062-I0460.
+'''July 6'''
-[[File:Jul.png]]
+Digestions and ligations :)
+'''July 8'''
+Colony PCR from the ligations made on July the 6th.
+'''July 11'''
+More digestions and subsequent ligations.
+We also created and isolated the following parts: K182102-C0061-B0015 & R0062-I0460.
 '''July 13'''
-BioBrick parts were digested using different combinations of restriction enzymes, ligated and run on a gel.
+Colony PCR from the ligations made on July the 11th.
+BioBrick parts were digested using different combinations of restriction enzymes and run on a gel.
+'''July 14'''
+Ligations were performed using the digestions from the 13th.
+'''July 15'''
+Colony pcr to verify the ligations of the 14th and subsequently liquid cultures of the positive colonies were made.
+We also created and isolated the following parts: K318511-S01003 & K182102-C0061-B0015-R0062-I0460 & R0040-K082014 &
+F2621-K082014-F2621 & F2621-E0422-F2621-K082014 & R0040-E0422 & F2621-K082014-F2621-E0422 & E0422-F2621-K082014 & & F2621-E0422-F2621-I0460 & R0040-K082014
+'''July 20'''
+Digestions, ligations, transformations.
+'''July 22'''
+PCR screen of the ligations from July 20th.
+'''July 23'''
+Digestions and ligations.
+'''July 25'''
+Transformations of the ligations from the 23rd.
+'''July 26'''
+We created and isolated the following parts: K182102-c0061-B0015 & R0062-I0460 & F2621-K082014 & F2621-I0460.
+More digestions, ligations and transformations.
+'''July 27'''
+Colony pcr to verify yesterday’s ligations.
-'''July 18'''
+'''July 28'''
-Gel extracion of the gel run on July 13th was performed. Liquid cultures were made.
+Digestions, ligations, transformations.
+'''July 29'''
-'''July 19'''
+Colony PCR and making liquid cultures
-Yesterday's liquid cultures were miniprepped.
+Also making new digestions and ligations.
 '''July 30'''
-Inoculated 500 mL erlenmeyers with 100 ml LB + 100 ug/ul. ''E. coli'' was grown overnight on LB + amp plates and directly used as inoculum. All cultures were done in duplo. ''E. coli'' strains used were:
+Isolating the parts from the liquid cultures made on the 29th.
+Besides that, we also inoculated 500 mL erlenmeyers with 100 ml LB + 100 ug/ul. ''E. coli'' was grown overnight on LB + amp plates and directly used as inoculum. All cultures were done in duplo. ''E. coli'' strains used were:
 ptet-GFP
 R0062-GFP