Team:USTC/Notebook/5 17

Modeling

Everybody

Date: May 15th

Recorder: Kaiyue Ma

Put the other two tubes in shaker for longer cultivating.

Date: May 16th

Recorder: Kaiyue Ma

Take all the four tubes(two in the shaker and two in the fridge), use the vortex to resuspend the bacteria, and mesure the intensity of each tube.

Use LB medium for zero calibration.

OD	No.1	No.2	No.3	No.4
Excitation peak: 584 nm	2.5 ABS	3.0 ABS	3.3 ABS	3.1 ABS
Emission peak: 607 nm	2.0 ABS	2.6 ABS	2.8 ABS	2.8 ABS

As the data is not ideally parallel, more experiments are needed.

Colony picking

Add 1.7 μL chloramphenicol (100 mg/mL) in 5 mL LB liquid medium (5 mL*4).

Pick the colnes from the original plate to four test tubes.

Cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.

Date: May 17th

Recorder: Yinchenguang Lv

Preparation of GuHCl solution

prepared 200 mL buffer solution for the simulating experiment of toxicity measuring.

The concentration of the buffer is 0.5 mol/L. Here are the ingredients:

component volume Disodium hydrogen phosphate dodecahydrate: 35.835 g Sodium dihydrogen phosphate dihydrate: 15.63 g

Diluted into a 200 mL solution.

Preparation of liquid medium

Chengle Zhang prepared 500 mL YPD liquid medium for yeasts last night. Here are the ingredians:

component proportion Yeast extract: 1% peptone：2% D-glucose： 2%

Date: May 18th

Recorder: Kaiyue Ma

As the first tube from left is far from expectation, we decide to cultivate it longer while store the rest in the fridge.

Date: May 19th

Recorder: Kaiyue Ma

Take all the four tubes, use the vortex to resuspend the bacteria, and mesure the intensity of each tube.

Use LB medium for zero calibration.

OD	No.1	No.2	No.3	No.4
Excitation peak: 584 nm	3.4 ABS	3.3 ABS	3.4 ABS	3.3 ABS
Emission peak: 607 nm	2.8 ABS	2.7 ABS	2.8 ABS	2.8 ABS

Date: May 24th

Recorder: Ya Jiang

1.The purposes

Add poly A to the 3' termination of DNA sequence and test whether the stability of corresponding mRNA will be enhanced.

2.The principles

it is supposed that poly A in the 3' termination of mRNA will increase its stablilty, but how many adenine residuals are efficient remains obsicure. In the pre-experiment, we use the part BBa_J04450 which contains a RFP sequence. Consequently, we can test the stability of mRNA by detect fluorescence intensity. Red/ET recombineering system is adopted to edit the DNA sequence and construct plasmids.

3.The materials and apparatus

(1)competent E·coil BL21 100 μL
(2)J04450-pSB1C3 plasmid 2 μL（50 pg/μL） (3)recombineering kits bought from Vazyme company(http://www.vazyme.com/) (4)spectrophotometer

4.procedures

4.1 PCR Amplification of Target DNA To clone any DNA fragment into a linearized vector using this kit, the insert fragment should be obtained by PCR using primers with an add-on of 15-20 bp homologous to either side of the restriction site that is used to linearize the vector. Therefore, the primer should be 5'-homologous-restriction site-poly A-restriction site-homologous-3'. 4.2 Preparation of Linearized Vector The linearized vector should be linearized by restrictiong endonuclease SAU3AI. 4.3 Recombination Procedure Set up the following reaction on ice in a 0.5 mL Eppendorf tube by mixing the following reagents gently and then spin down briefly to collect the reagents at the bottom of the tube.

column1	column2
Deionized water	up to 20 μL
5X CE MultiS Buffer	4 μL
lineratized vector	x ng
PCR products	x ng

x=[0.02*bp]ng(0.03 pmol)

Incubate the reactions at 37°C for 30 minutes, and then transfer tubes to ice and incubate on ice for 5 minutes. 4.4 Proceed with transformation The reaction can also be stored at -20°C for later transformation. 4.5 Transformation 1. Thaw one vial of frozen 200 μL competent cells on ice. Tap the tube gently to ensure that the cells are suspended. 2. Add 20 μL of reaction mixture to the competent cells. Tap the tube gently and incubate the tube on ice for 30 minutes. 3. Heat shock the cells by placing them in 42°C water bath for 45-90 seconds and then place the tube on ice for 2 minutes. 4. Add 900 ul of SOC medium or LB culture to the cells and then incubate the cells on a shaker set at 200 rpm at 37°C for 60 minutes. 5. Centrifuge the cell down at 5000 rpm for 5 minutes and then remove and discard about 100 μl of medium. Gently suspend the cells by tapping the tube. 6. Incubate the plates overnight at 37°C.

Date: May 28th Recorder：Chengle Zhang

Pre-test for Toxicity Testing of GuHCl to Yeast W303

Experimental materials: YPD culture medium 400 mL(made by ourselves) Yeast W303(made by ourselves)

Procedure: 1. Subpackage the YPD medium into 20 test tubes(5 mL per tube) and into 2 erlenmeyer flasks(95 mL each). 2. Pick the colonies of Yeast W303 into 2 test tubes,then cultivate them at 30 degree centigrade and shake at 250 rpm/min 24h. 3. Store medium of the left tubes and flasks at 4°C.

Date: 5/28 by Yinchenguang Lyu prepared 10ml 2mol/L GuHCl solution and 1500ml YPD midium.

Date: May 28th Recorder：Chengle Zhang

Pre-test for Toxicity Testing of GuHCl to Yeast W303

Experimental materials: YPD culture medium 400 mL(made by ourselves) Yeast W303(made by ourselves)

Procedure: 1. Subpackage the YPD medium into 20 test tubes(5 mL per tube) and into 2 erlenmeyer flasks(95 mL each). 2. Pick the colonies of Yeast W303 into 2 test tubes,then cultivate them at 30 degree centigrade and shake at 250 rpm/min 24h. 3. Store medium of the left tubes and flasks at 4°C.

Date: May 29th by Chengle Zhang

There are some precipitations in the two test tubes with yeast.

Yeast Growth Curve Measurement 1

Procedure: 1. 24 h later, put the medium with yeast in the two tubes into two flasks mentioned above respectively. 2. Time from now, take 1 mL liquid from each of the two flasks and the original medium into EP tube respectively after specific time(shown in the table below). 3. Measure the OD data of each EP tube.

Results:

No.	Time point	Time/h	OD600 Sample1	OD600 Sample2	OD600 Average
1	9:15	0	0.176	0.158	0.167
2	10:45	1.5	0.185	0.184	0.184
3	12:15	3	0.617	0.558	0.588
4	13:35	4.33	0.680	0.691	0.686
5	15:15	6	2.180	3.180	2.680
6	17:15	8	4.128	3.704	3.916
7	19:35	10.33	5.230	5.280	5.255
8	20:48	11.55	6.910	6.870	6.890
9	22:00	12.75	6.650	6.620	6.635
10	23:00	13.75	8.330	7.750	8.040
11	0:00	14.75	8.502	8.619	8.560
12	1:00	15.75	9.464	8.125	8.794
13	2:01	16.77	9.529	9.776	9.652
14	3:00	17.75	10.374	9.503	9.938
15	4:00	18.75	10.560	11.104	10.832
16	5:03	19.8	10.672	10.560	10.616
17	6:00	20.75	10.912	10.752	10.832