Notebook

Grand Meterial

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LB Culture(100mL):
Trytone 1g
Extract 0.5g
NaCl 1g
(if solid) Agar 1.5g

8.3 Experimental Record

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Topic：Plasmid Amplification
Researcher: Xinyi Zhao & Yuxuan Wu
Material：
plasmid——pQCXIP-mCherry (A+)
competent cell——Tran10
Methods/protocol：
Mix 100ng-500ng pQCXIP-mCherry with 100μl Tran10
Ice bath 30mins
Heat stimulation 42℃，90s
Ice bath 2-3 mins
        Spread-plate on A+ medium
Shaker, 37°C 200rpm，18h
 
[Design of primer: basic instructions]
Take pQXCIP & MO2 as an example
1.15bp overlap with MO2
2.include cleavage sites
3.18bp overlap
 
Wu: Example plasmid and primers

8.4. Discussion record

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    Topic: Discussion about following steps and lactoferrin
    Reseacher:Xinyi Zhao & Yuxuan Wu
1.Buy lactoferrin and verify that it has functions to retrain bacteria’s growth
2.Search for method of bacteria counting
3.Two ways to free the bacteria from the effect of LTF: over-expression of transferrin such as Feo family; add ferric or ferrous ions to the medium
4.Use Coomassie blue stain or western blot to analyze

8.17 Experiment record

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    Topic: preliminary experiment
    Reseacher: Xinyi Zhao & Fangming Liu & Zirui Fang & Zijun Yin & Yuxuan Wu
Operation 1: Make LB medium, using autoclave to sterilization.
Operation 2: Do mock electrophoresis using retrovirus.
Operation 3：Calculate the concentrate, use ferric chloride to prepare ferric ions solution.
Operation 4: Bacteria liquid inoculate.

8.18 Experiment record

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    Topic: preliminary experiment
    Reseacher: Zirui Fang & Yuxuan Wu
Operation 1: adjust PH value of ferric solution by using sodium hydroxide.
Result: Fail, sediment appeared.
Reason: sodium hydroxide was too concentrated. Carbonate or bicarbonate solution should be used.
 
2.Prepare mole salt solution（MS）.Experiment aimed as the effect of LTF effect and ferric ion impact on the effect.
Groups in experiment：/N，LTF，MS，LTF+MS，three repetition for each group
Result：The OD value for each group didn’t have much difference.However, bacteria has obviously differnce in appearance. In the group of adding LTF, dead bacteria clinged to the tubes’ wall. Mixing operation led to the result of OD values. MS doesn’t have significant effect.

8.21 Experimental record

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Topic: cDNA preparation
Researcher: Fangming Liu
Main：Use the high-expressed ltf mRNA （4uL/sample） of nasal mucosa cell, offered by the lab,prepare the cDNA contains LTF sequece. Later design primers for amplification of the cDNA.
Material：
1.mRNA sample x3
2.SuperRT retrotranscription kit
（1）SuperRT Reverse Transcriptase
（2）SuperRT Buffer
（3）dNTP mix
（4）Primer mix
（5）RNase free water
Steps：
1.Construct a reaction system（19uL/sample）
2.Put into Thermal Cycler （50min at 42C+ 10min at 85C，4C standby）
3.Using Ultramicrospectrophotometer to detect DNA concentration and quality.
4.Preserve in -20°C environment.
Later steps：
1.Design primers
2.Design Sanger sequencing primer
3.PCR, amplifcation of each isoforms.
4.Design of full sequence Sanger sequencing primers
5.Construct plasmid
Sample 1（Rs0004R）：1014ng/uL
Sample 2（875811-pl）：994ng/uL
Sample 3（875811-GT）：923ng/uL
（260/280 and 230/260 conform to single strand DNA standard）

See engineering and experiment part.

9.16 experimental & discussion record

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Experimental design：
Use OD600=1.3 bacteria liquid to construct expression system. （2mL bacteria liquid）
groups：
16°C：
1）IPTG 1:500
2）IPTG 1:1000
3）IPTG 1:2000
25°C：
4）IPTG 1:500
5）IPTG 1:1000
6）IPTG 1:2000
37°C：
7）IPTG 1:500
8）IPTG 1:1000
9）IPTG 1:2000
Fe2+ （T=25C，IPTG 1:1000）
10）20x dilution Fe2+
11）5x dilution Fe2+
12）no dilution
Fe3+ （T=25C，IPTG 1:1000）
13）20x dilution Fe3+
14）5x dilution Fe3+
15）no dilution
Without iptg（T=25C）：
16）standard concentration of Fe2+
17）standard concentration of Fe3+
18）controling group（E）：bacteria liquid（37C）

Specifically, based on the experiment of last time, the next step of experiment is carried. 16°C，IPTG1:1000, ferric ion solution without adding chelating agent or adjusting PH value, because the PH value of the solution will not have huge impact on the whole system.
a）Fe2+（9.9mM）
19）100x dilution
20）50x dilution
21）20x dilution
22）10x dilution
23）5x dilution
24）standard
25）5x concentration
26）10x concentration
27）20x concentration
28）50x concentration
29）100x concentration
b）Fe3+（9.9mM）
same as Fe2+
（30-40）
 c）other（16°C） without IPTG
41）controling group2
42）Fe2+ 20x dilution only
43）Fe2+ 5x dilution only
44）Fe2+ 1x
45）Fe2+ 5x concentration
46）Fe2+ 20x concentration
Fe3+ （47-51、53）
The same as Fe2+
Add 10x dilution（50）
53) 20x dilution
52） 25°C controling group
In summary, there are 102 groups,additional with 10 MoE group.

（15-1）· 8= 112
 
material：
system：LB medium（Kanamycin+） 4mL/group
IPTG：
1:500 8uL/group
1:1000 4uL/group
1: 2000 2uL/group
Fe ions solution：1x group has final concentration of 0.1386mM
2mL system for 30mg LTF saturated concentration (27uL original solution) (1x)
1x for 54uL original solution in 4mL system
Bovine LTF Standard
 
Result:
15:05 Inoculation in 250mL LB（K+）with 5mL ECS (1:50)
Kanamycin 1:1000
17:00 OD600=0.6
17:35 OD600=0.8
18:12 OD600=1
19:15 OD600=1.2

The western blot and Coomassie blue stain: