Difference between revisions of "Team:USTC/Notebook"

Medium preparation

Prepare and keep a part of solid medium and broth in the refrigerator as a backup.

Cell culture

Culture the cell,TOP10, with LB liquid solution overnight.

Design protocol

1. Take the solid medium, heat it until liquid. Then make two plate of solid medium, evenly. Cool it to the room temperature.
2. prepare antibiotic solution with concentration gradient
   a. prepare antibiotic(neomycin) solution with ddH2O at 50mg/ml as mother solution 1. And dilute it to 10% concertration(5mg/ml) with ddH2O as mother solution 2. Then prepare 8 parts of solution as this:

Keep them in the 70℃ water bath. Then add 800uL liquid solid medium into each Ep tubes. Transfer them into 50℃ water bath.

1. Cut 4 hole in each plate of solid medium, whose diameter is at 8mm.
2. Take 5mL culture into the 2 Ep tubes. Centrifuge it at twice. Then add liquid solid medium at 50℃, each 800uL, dissolve evenly.
3. Take 200uL top10 solution into 8 Ep tubes each at 50℃. Then add the mixed solution into the hole at once until the hole is added up.

Results: the raw results showed differerent characteristics of chemotaxis based on distinct antibiotic concentration.

Analysis: 其中3号有最明显的趋化结果，说明这个趋化结果并不是完全正相关或负相关的。但是结论可能是存疑的，过量的加入固体培养基会导致可能的菌液溢出从而形成如此的趋化形状。因此实验配方需要进行更改。

今天在延续着昨天的实验思路，利用灭菌的1mL枪头打洞，然后将含抗培养基和菌液混合，打入洞中，待凝固后放入恒温箱培养。
实验结果:这次并没有长出相应的环带。有可能是由于在放入之前被紫外灯照射了的缘故导致的细菌死亡。但是同时也有可能是由于孔洞较深，使得大肠杆菌没有办法从边缘逃逸。

前一天的培养基里头出现了E.coli,但是伴随着杂菌。
于是重复了7月6日的实验，此次打洞是，为了方便控制液体体积，这会使用的是灭菌的200uL枪头打洞。不过由于体积过小，导致仍有一部分液体溢出。

发现7月7日的结果与7月5日结果有差别，并且各孔的趋化效果呈振荡形式，并且环的效果不是很明显。

analyse：形成环的一部分孔洞是存在着溢出情况的，某种程度上印证的之前关于溢出的猜测。
可能E.coli的运动能力不是很强，引起环的明显程度不够，或者是由于抗生素的浓度过高，而杀死或抑制了细菌。后面实验还需要重新审核实验方案，并且需要克服由于加样无法精确掌握体积的问题。

接下来的思路：可以考虑利用含有抗性的菌来做趋化实验，但是首先要求该种抗性仅仅是抗生素作用靶点的改变，而不是分解或者是抗生素失活的方式，以免改变抗生素的局部浓度。

转化

今天转化的是来自刘玉婷实验室的TG1感受态细胞，感受态细胞是由刘玉婷与张启钧提前完成的，主要目的是为了检测：

• 感受态细胞是否高效
• 感受态细胞是否污染

实验流程：

1. 冰上放置感受态细胞2~3min，一共4管
2. 加入5ul检测质粒，其中3管有100ul感受态细胞两支，200ul感受态细胞两支，其中100ul的感受态一支加入5ul质粒，一支加入1ul质粒，在冰上放置30min
3. 在42℃水浴锅中热激45s
4. 冰上放置3min
5. 加入900ulLB培养基培养55min
6. 离心12000g 15s，放弃上清液，加入200ulLB培养基
7. 涂板，板带有氨苄抗性
8. 培养20h观察实验结果

绿脓杆菌接菌

从金范老师那里拿来的绿脓杆菌生长完整，选取了其中的三个菌落挑单，发现绿脓杆菌单菌落相比大肠杆菌较大，培养基有异味。取5mL LB培养基直接将挑好的细菌用枪头打在其中培养16h次日以备使用。

冻存绿脓杆菌

绿脓杆菌需要冻存，冻存的最后甘油体积分数为15%

灭菌时配好的50%甘油进行灭菌，因为根据小木虫的经验帖，100%体积的甘油不宜直接灭菌

甘油的密度为：1.26g/mL

50%甘油（50mL）配方为：

• 25mL 蒸馏水
• 25mL（31.5g）甘油

混匀之后即可灭菌，冻存管同时灭菌

15%冻存甘油管的配方为（1mL）：

• 700ul 培养菌液
• 300ul 15%甘油溶液

冻存于-40℃冰箱中，备用

涂板绿脓杆菌

早上：抽取培养过的绿脓杆菌取100ul加入LB固体培养基中进行涂板

结果发现，细菌密度过浓，没有单菌落形成

晚上：抽取培养的绿脓杆菌，进行1：4的培养基稀释，之后取100ul加入到LB固体培养基中涂板，过夜培养

转化

转化材料是来自洪炯老师的XL21 Gold感受态细胞，和昨日刘玉婷制备的TG1感受态细胞，希望进一步证明感受态细胞没有被污染，以及扩增pSBC1C3质粒，和T7启动子，tRNA part

实验流程：

1. 冰上放置感受态细胞3min，一共4管
2. 稀释parts（T7启动子：2013年Kit5的6N孔，tRNA：2015年Kit5的21I孔），加入10ul灭菌过的蒸馏水，稀释混匀。
3. 加入1ul检测T7启动子与tRNA的parts至XL21 gold感受态细胞，加入5ulpSB1C3至XL21 Gold感受态细胞，在冰上放置30min
4. 在42℃水浴锅中热激55s
5. 冰上放置3min
6. 加入到5mL的氯霉素抗性的液体培养基。
7. 培养12h明日观察结果（21：00完成）

1st Colonial PCR for COMPX

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl COMPX-F Primer
• 1μl COMPX-R Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl TOP10 Solution
• 12.35μl ddH2O

Renaturation at 45℃, extend for 1 min.

1st Electrophoresis for Colonical PCR

No band except marker resulted due to Gelred past due.

重新转化实验

氯霉素的浓度昨天有误，今天重新进行配置，再养菌

重新制备感受态细胞

感受态细胞用Amp进行空白测试

重新对绿脓杆菌涂板

涂板稀释浓度增加到1:10与1:15，因为绿脓杆菌浓度过高，单菌落太少，不宜储存

2nd Colonial PCR for COMPX

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl COMPX-F Primer
• 1μl COMPX-R Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl TOP10 Solution
• 12.35μl ddH2O

Renaturation temperature varies from 50℃ to 60℃, extend for 1 min.

2nd Electrophoresis for Colonical PCR

Add Gelred in loading buffer. The band of PCR product appears in all samples.

结果如下图所示：

意外出现问题，由于胶用水去溶解了，导致Marker都没有跑开

该结果则较好的显示出了菌落PCR成功的p出了COMPX蛋白

教训：

• 误用琼脂
• 误用水区溶解胶
• 电压需要调整回90V，30min

3rd Colonial PCR for COMPX

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl COMPX-F Primer
• 1μl COMPX-R Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl TOP10 Solution
• 12.35μl ddH2O

Renaturation at 55℃, extend for 1 min.

3rd Electrophoresis for Colonical PCR

No results again.

1st Transformation

1 Add 1μl BBa K1492002 or 5μl pSB1C3 plasmid to competent cells.

2 Heat at 42℃ for 45 s by mistake.

3 Incubate on ice for 30 min.

4 Heat at 42℃ for 45 s.

5 Incubate on ice for 3 min.

6 Add 900 μl LB medium, incubate at 37℃ for 1 h.

7 Centrifuge and discard 85 μl LB medium.

8 Resuspend cells and add 100 μl to LB medium with chloramphenicol. Incubate at 37℃.

4th Colonial PCR for COMPX

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl COMPX-F Primer
• 1μl COMPX-R Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl TOP10 Solution
• 12.35μl ddH2O

Renaturation at 55℃, extend for 1 min. 2 tubes without Taq DNA Polymerase as blank control.

4th Electrophoresis for Colonical PCR

Bands are predicted except one. Maybe caused by accident. PCR product keeped for later use.

1st Plasmid Extraction for BBa K1492002 & pSB1C3

1 Centifuge 5 ml bacterium solution, few sediment getted.

2 Add 250 μl Buffer P1, resuspend cells.

3 Add 250 μl Buffer P2, mix well, 4 min's standing.

4 Add 350 μl Buffer P3, mix well.

5 13.4 rpm centifuge 10 min, move all supernatant to adsorption column, 11.6 rpm centifuge 30 s, discard filtrate.

6 Add 500 μl Wash Solution, 11.6 rpm centifuge 30 s, discard filtrate. Repeat once.

7 11.6 rpm centifuge 1 min.

8 Add 50 μl ddH2O, 2 min's standing, 11.6 rpm centifuge 1 min.

1st Electrophoresis for Plasmid Extraction

No result. May due to high concentration of chloramphenicol, bacteria grow very slow. As there is few bacteria, no plasmid getted. Bacterium solution dilute 1:1 with LB medium.

绿脓杆菌划线

大约在傍晚7点左右，利用划线法在LB平板上培养绿脓杆菌。一块是从昨天的培养的培养基里分出来的，另一块是从菌液中沾取划线的。

10点离开前，看了一次，从培养基分出来的那一块平板已经有菌落生成了。

The steak-plate of Pseudomonas aeruginosa

By using the steak-plate method, we successful separated the single conlony of P. aeruginosa.
\(^o^)/~！

2nd Plasmid Extraction for BBa K1492002 & pSB1C3

1 Centifuge 4.5 ml bacterium solution.

2 Add 250 μl Buffer P1, resuspend cells.

3 Add 250 μl Buffer P2, mix well, 4 min's standing.

4 Add 350 μl Buffer P3, mix well.

5 13.4 rpm centifuge 10 min, move all supernatant to adsorption column, 11.6 rpm centifuge 30 s, discard filtrate.

6 Add 500 μl Wash Solution, 11.6 rpm centifuge 30 s, discard filtrate. Repeat once.

7 11.6 rpm centifuge 1 min.

8 Add 30 μl ddH2O, 2 min's standing, 11.6 rpm centifuge 1 min.

2nd Electrophoresis for Plasmid Extraction

Successfully extracted tRNA plasmid. Failed to extract pSB1C3 plasmid, cause culture is contaminated.
Result:

tRNA 114ng/μl

1st PCR for tRNA

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl tRNA-F2 Primer
• 1μl tRNA-R Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl tRNA plasmid
• 12.35μl ddH2O

Renaturation at 50℃, extend for 1:30.

1st Electrophoresis for tRNA PCR

No result.

Transformation of Four Parts of Plate 4

1. material:
   competent cell TG1 from the lab 319
   4 parts of the plate 4(pSB1A2)
   No.1:BBa_B0034 No.2:BBa_C0051
   No.3:BBa_R0051 No.4:BBa_E0040
2. the procedure
   (1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
   (2)Absorb 1 uL or 2 uL plasmid and mix it with bacteria solution thoroughly.
   (3)Put the tubes on the ice about 30 mins.
   (4)Make a heat shock at 42 degree centigrade about 60 sec
   (5)Put the tubes on the ice about 3 mins again.
   (6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
   (7)Centrifuge them at 4,000xg about 15 sec and we will se sediment in the tubes.
   (8)Discard the supernatant liquid and leave about 100ul medium.
   (9)Coat plate: add 100ul solution in a plate

The Results of Transformation of Jul 13th

transformations of three parts are successful:
No.1 B0034
No.2 C0051
No.4 E0040
transformation of one part is failing:
No.3 R0051

Plasmid Extraction of pSB1C3

the plasmis:pSB1C3

the procedure:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 1 min and centrifuge about 1 min at 12,000xg.
PS:this protocol is from lab 319

the concentration :11.0ng/ul
PS:the concentration of the control plasmid is 136.0 ng/ul,so we guess that maybe the concentration of bacterium solution is low.Also we guess that it's caused by the missing of spreading.

Transformation of Three Parts and One Plasmid

1. the plasmid going to be transformed:
   No.1 BBa_R0062(plate 2;pSB1C3)
   No.2 BBa_B0015(plate 3;pSB1C3)
   No.3 BBa_R0051(plate 4;pSB1A2)
   No.4 pSB1C3
2. the procedure:
   note:add 1 ul R0062 ,1 ul B0015,2 ul R0051,
   2 ul pSB1C3 to 100 ul competent cells respectively

(1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
(2)Absorb 1 uL or 2 uL plasmid and mix it with bacteria solution thoroughly.
(3)Put the tubes on the ice about 30 mins.
(4)Make a heat shock at 42 degree centigrade about 60 sec
(5)Put the tubes on the ice about 3 mins again.
(6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
(7)Centrifuge them at 4,000xg about 15 sec and we will se sediment in the tubes.
(8)Discard the supernatant liquid and leave about 100ul medium.
(9)Coat plate: add 100ul solution in a plate

Plasmid Extraction of Three Parts of Plate 4

the procedure is the same as before
the concentration :
B0034 157.6ng/ul, 113.7ng/ul;
C0051 171.7ng/ul, 194.6ng/ul;
E0040 278.5ng/ul, 228.2ng/ul

Results of transformation

R0051 no colony
R0062 have single colony
B0015 have single colony
pSB1C3 have single colony and the colony is red

Plasmid Extraction Results

This is extraction was for previous transformation.

• pSB1C3(1): 108ng/uL
• pSB1C3(2): 157ng/uL
• B0015(1): 19ng/uL
• B0015(2): 93ng/uL

Prepared for further use.

Colonical PCR for micF and SoxS

Primers produced from Sangon Biotech Co.,Ltd.

For micF, primers including micF-luxI-R, micF-F, protocol as following:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl micF-luxI-R Primer
• 1μl micF-F Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl Top10(K-12) Strain E. coli bacterial solution
• 12.35μl ddH2O

For SoxS, primers including SoxS-RBS-luxI-R, BB-SoxS-F, protocol illustrates as:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl SoxS-RBS-luxI-R Primer
• 1μl BB-SoxS-F Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl 1μl Top10(K-12) Strain E. coli bacterial solution
• 12.35μl ddH2O

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 7 min
4. 4 celcius degree for preserveation.

Characterization of Colonical PCR by Gel Electrophoresis

The result illustrated the success of PCR

Colonical PCR for OprF from Psudomonus aeruginosa

Primers produced from Sangon Biotech Co.,Ltd.

PCR protocol:

For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl OprF-F Primer
• 1μl OprF+Ter-R Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl Psudomonus aeruginosa (PAO1) bacterial solution
• 12.35μl ddH2O

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 7 min
4. 4 celcius degree for preserveation.

Plasmid Extraction of SCVE

SCVE synthesized from Sangon Biotech Co.,Ltd.

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 1 min and centrifuge about 1 min at 12,000xg.

Transformation for CRISPR

1.材料：competent cell TG1 from the lab 319，4Mparts of the plate 4(pSB1A2)

2.编号：1:BBa_B0034 2:BBa_C0051

3:BBa_R0051 4:BBa_E0040

3.步骤：

（1）将感受态细胞置于冰上，孵化5分钟；

（2）取1ul 含代转parts的plasmid与感受态细胞混合，轻轻混匀；

（3）冰上放置30min，中间轻摇数次，防止菌体沉淀；

（4）42.0度水浴静置热激90s（这个时间控制很重要），立即放入冰上冷却2-3min；

（5）加入900ul LB(不含抗生素)培养基，混匀；

（6）37度振荡培养1h；

（7）12，000rpm 离心15s，用移液器小心吸出850ul上清丢弃后，保留50ul重悬菌液;

（8）用移液器混匀菌液，均匀涂布于含抗生素（Cl 200ml LB:50ul 100ng/ul Cl）的平板上，放入37度培养箱倒置培养过夜。

Characterization of SCVE and OprF by Gel Electrophoresis

Only 1/4 PCR results successfully duplicated results, plasimid extraction from SCVE showed well.

Second PCR for OprF didn't work well:(

Digestion of E0040(EGFP)

Enzyme from Thermo Scientific FastDigest Enzyme with Green Buffer

Protocol as following:

• Nuclease-free water: 15ul
• E0040: 1ul
• 10* FastDigest Green Buffer: 2ul
• EcoRI: 1ul
• SpeI: 1ul

We are trying to figure out the best conditions of our experiment and whether we need loading buffer

PCR of E0040(EGFP)

Primers produced from Sangon Biotech Co.,Ltd.

For E0040, primers including E0040-F, R0051-E0040-R, protocol as following:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl E0040-F Primer
• 1μl R0051-E0040-R Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl plasmid(pSB1C3) containing E0040
• 12.35μl ddH2O

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 7 min
4. 4 celcius degree for preserveation.

Attention: R0051-E0040-R Primer is not appropriate because of only 8 complementary base pairs.

Characterization of E0040 from Digestion and PCR

Result illustrated that We do need loading buffer, and 5 min do work!

PCR showed more concentration of E0040!

Gel Extraction

Preparation:

1. Check out whether ethyl alcohol is added into Wash Solution.
2. Check out whether Buffer B2 has sediment.
3. Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

1. Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
2. Measure out the weight of tubes of gel.
3. Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
4. Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
5. After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
6. Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
7. Do the step 5 again.
8. Centrifuge the empty columns at 9000xg about 1min.
9. Open the columns and air them about 10 mins.
10. Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
11. Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

Final OD results:

Perseved for further use.

Transfofmation of Three Parts

Protocol as following

• 100μl (three tubes)competent cells
• C0062(pSB1C3)
• R0010(pSB1C3)
• C0061(pSB1C3)
• LB without antibiotic
• LB medium with Amp/CmR

Plasmid Extraction of E0040

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

Results

Colonical PCR for OprF from Psudomonus aeruginosa

PCR protocol:
For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl OprF-F Primer
• 1μl OprF+Ter-R Primer
• 0.5μl Taq DNA Polymerase(add more 0.25ul then previous protocol)
• 2μl MgCl2
• 1μl Psudomonus aeruginosa (PAO1) bacterial solution
• 12.35μl ddH2O

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 7 min
4. 4 celcius degree for preserveation.

Digestion of E0040(EGFP)

Enzyme from Thermo Scientific FastDigest Enzyme with Green Buffer

Protocol as following:

• Nuclease-free water: 15ul
• E0040: 1ul
• 10* FastDigest Green Buffer: 2ul
• EcoRI: 1ul
• SpeI: 1ul

Characterization of OprF and E0040

酶连效果不是很好，基本建议是改善昨日胶回收的条带进行PCR扩增，之前引物设计担心不是很好，但是看样子没有其他位点被p了出来；
OprF的PCR依然是在相同条件下只有一个条带p了出来，比较异常；此外引物二聚体较多，因此对此批进行了胶回收，以备后续使用。

Plasmid Extraction of Cas9(K1218011)

The bacteria were cultured at 9 am from successfully transformed bacteria.

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

Characterization of Cas9

转化成功，明天写实验结果讨论

Digestion of K1218011(Cas9)

Enzyme from Thermo Scientific FastDigest Enzyme with Green Buffer

Protocol as following:

• Nuclease-free water: 15ul
• K1218011: 1ul
• 10* FastDigest Green Buffer: 2ul
• EcoRI: 1ul
• SpeI: 1ul

Incubate into 37 degree celcius, 2h

Digestion of SCVE

Enzyme from Thermo Scientific FastDigest Enzyme with Green Buffer

Protocol as following:

• Nuclease-free water: 16ul
• SCVE: 1ul
• 10* FastDigest Green Buffer: 2ul
• SmaI: 1ul

Incubate into 37 degree celcius, 2h

Colonical PCR for OprF from *Psudomonus aeruginosa, micF and SoxS from K-12

PCR protocol:

For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl OprF-F Primer
• 1μl OprF+Ter-R Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl Psudomonus aeruginosa (PAO1) bacterial solution
• 12.35μl ddH2O

For micF, primers including micF-luxI-R, micF-F, protocol as following:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl micF-luxI-R Primer
• 1μl micF-F Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl Top10(K-12) Strain E. coli bacterial solution
• 12.35μl ddH2O

For SoxS, primers including SoxS-RBS-luxI-R, BB-SoxS-F, protocol illustrates as:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl SoxS-RBS-luxI-R Primer
• 1μl BB-SoxS-F Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl 1μl Top10(K-12) Strain E. coli bacterial solution
• 12.35μl ddH2O

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 7 min
4. 4 celcius degree for preserveation.

Praparation for Competent Cell

protocol as the following:

1. Pick out two single colonies(TG1) and cultivate bacteria in 5mL LB media at 37 degree centigrade about 12 hours with shocking.
2. Take 5mL nutrient solution into 100 mL media and cultivate bacteria at 37 degree centigrade about one hour and a half with shocking till the OD660 reaches 0.4-0.5.
3. Absorb 50 mL nutrient solution into EP tubes with ice-bath about 10 mins.
4. Centrifuge the tubes at 4100xg at 4 degree centigrade about 10mins and discard the supernatant liquid.
5. Utilize 30 mL 0.1M calcium chloride to suspend bacteria.
6. Centrifuge the tubes at 4100xg at 4 degree centigrade about 10 mins and discard the supernatant liquid.
7. Utilize 2 mL 0.1M calcium chloride with 15% glycerol to suspend bacteria and then ready for transformation quickly or restore at -70 degree centigrade.

PS:dilute the solution by adding 100 mL LB media after first test.

Negative Chemotaxis Characterization

term 1, material:

• LB solid medium plates x4
• 1g/ml Chloramphenicol
• E.coli Top10
• filter paper

Plasmid Extraction of EmrE and ArcB

done by Wang Luyao

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

the concentration as following
EmrE 70.2 ng/ul 92.3 ng/ul
ArcB 62.6 ng/ul 95.2 ng/ul

Transformation of R0051 and C0061

the source of parts:
No.1 R0051 2014 kit plate 4
No.2 R0051 2014 kit plate 4
No.3 control
No.4 C0061 2015 kit plate 4
No.5 C0061 2015 kit plate 4
No.6 C0061 2014 kit plate 4

No.1 and No.4 the competent cell is from lab319
No.2 and No.5 the competent cell is made by us (firt)
No.3 and No.6 the competent cell is made by us (second)

the protocol as following
(1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
(2)Absorb 1 uL or 2 uL plasmid and mix it with bacteria solution thoroughly.
(3)Put the tubes on the ice about 30 mins.
(4)Make a heat shock at 42 degree centigrade about 60 sec
(5)Put the tubes on the ice about 3 mins again.
(6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
(7)Centrifuge them at 4,000xg about 15 sec and we will se sediment in the tubes.
(8)Discard the supernatant liquid and leave about 100ul medium.
(9)Coat plate: add 100ul solution in a plate

PCR of OrpF

material:

• 13 uL Taq Master Mix
• 11 uL dd water
• 1 uL PAO1 bacterial
• 0.5 uL primer Fd(OrpF-F)
• 0.5 uL primer Rv(OrpF+Ter-R)
  PS:Taq Master Mix is from lab 319 ,and this reagent is used for bacterial PCR.

Renaturation at 50℃, extend for 1:10

PCR of OprF

material:
No.1 and No.2:

• 6.5 uL Taq Master Mix
• 1 uL PAO1 bacterial solution
• 4.5 uL dd water
• 0.5 uL OprF-F
• 0.5 uL OprF+Ter-R

No.3 and No.4:

• 6.5 uL Taq Master Mix
• 4 uL OprF PCR product
• 1.5 uL dd water
• 0.5 uL OprF-F
• 0.5 uL OprF+Ter-R

PS:Taq Master Mix is from lab 319 ,and this reagent is used for bacterial PCR.

Renaturation at 55℃, extend for 1:10

OprF for PCR and gel electrophoresisOprF

analyze: the length of the product is

Preparation of Semi-Solid LB Medium

In order to characterization the movement of TOP10, semi-solid LB medium preparation is indispensable in this research.

(500mL Protocol)

Compared with solid LB medium, containing 1.5% agar in there.

Plasmid Extraction C0051,B0015 and R0062

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

the concentration as following
C0015:171.7 ng/uL
B0015:93 ng/uL
R0062:144.3 ng/uL

PCR of C0051,B0015 and R0062

material:
C0051 plasmid(171.7 ng/uL)
B0015 plasmid(93 ng/uL)
R0062 plasmid(144.3 ng/uL)
all the solutions are diluted 10-folds

Primers produced from Sangon Biotech Co.,Ltd.

No.1 and No.2 for C0051(803bp)
Protocol as this:

• 2.5 μl Taq buffer with KCl
• 2.5 μl dNTPs
• 0.75 μl C0051-F Primer
• 0.75 μl C0051-B0015-R(I) Primer
• 0.5 μl Taq DNA Polymerase
• 1.0 μl MgCl2
• 2 uL C0051 solution
• 15 μl ddH2O

No.3 and No.4 for B0015(142bp)
Protocol as this:

• 2.5 μl Taq buffer with KCl
• 2.5 μl dNTPs
• 0.75 μl B0015-F Primer
• 0.75 μl B0015-R(I) Primer
• 0.5 μl Taq DNA Polymerase
• 1.0 μl MgCl2
• 3 uL C0051 solution
• 14 μl ddH2O

No.5 and No.6 for R0062(104bp)
Protocol as this:

• 2.5 μl Taq buffer with KCl
• 2.5 μl dNTPs
• 0.75 μl R0062-F Primer
• 0.75 μl R0062-C0051-R Primer
• 0.5 μl Taq DNA Polymerase
• 1.0 μl MgCl2
• 2 uL R0062 solution
• 15 μl ddH2O

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 2min(C0061)or 1min(B0015 and R0062)
4. 4 celcius degree for preserveation.

Preparation Semi-Solid M63 Medium

In order to characterization the movement of TOP10, semi-solid M63 medium preparation is indispensable in this research.

(500mL Protocol)

Colonical PCR for OprF from Psudomonus aeruginosa and SCVE from pUC57

PCR protocol:

For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl OprF-F Primer
• 1μl OprF+Ter-R Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl Psudomonus aeruginosa (PAO1) bacterial solution
• 12.35μl ddH2O

For SCVE, Primers produced from Sangon Biotech Co.,Ltd.

Protocol as this:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl SCVE-T-R Primer
• 1μl T7-RBS-SCVE-F Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl SCVE solution from plasmid extraction
• 12.35μl ddH2O

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 7 min
4. 4 celcius degree for preserveation.

Gel Electrophoresis of OprF, SCVE micF and SoxS

micF and SoxS

OprF and SCVE

Gel Extraction

Material including: OprF, SoxS, micF

Preparation:

1. Check out whether ethyl alcohol is added into Wash Solution.
2. Check out whether Buffer B2 has sediment.
3. Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

1. Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
2. Measure out the weight of tubes of gel.
3. Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
4. Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
5. After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
6. Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
7. Do the step 5 again.
8. Centrifuge the empty columns at 9000xg about 1min.
9. Open the columns and air them about 10 mins.
10. Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
11. Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

Plasmid Extraction of R0062

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

PCR for OprF and C0061

PCR protocol:

For OprF, primers including OprF-F, OprF+Ter-R, protocol as following:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl OprF-F Primer
• 1μl OprF+Ter-R Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl Psudomonus aeruginosa (PAO1) bacterial solution
• 12.35μl ddH2O

For SCVE, Primers produced from Sangon Biotech Co.,Ltd.

Protocol as this:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl C0061-F Primer
• 1μl C0061-B0015-R Primer
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1μl C0061 solution
• 12.35μl ddH2O

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 7 min
4. 4 celcius degree for preserveation.

Gel Extraction of C0062,B0015 and R0062

Material including: C0051,B0015 and R0062

Preparation:

1. Check out whether ethyl alcohol is added into Wash Solution.
2. Check out whether Buffer B2 has sediment.
3. Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

1. Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
2. Measure out the weight of tubes of gel.
3. Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
4. Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
5. After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
6. Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
7. Do the step 5 again.
8. Centrifuge the empty columns at 9000xg about 1min.
9. Open the columns and air them about 10 mins.
10. Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
11. Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

the concentration as following:
C0051:12.7 ng/uL
B0015:7.3 ng/uL
R0062:7.7 ng/uL

PCR of OprF

material:

• 13 uL Taq Master Mix
• 11 uL dd water
• 1 uL PAO1 bacterial
• 0.5 uL primer Fd(OrpF-F)
• 0.5 uL primer Rv(OrpF+Ter-R)

Renaturation at 55℃, extend for 2min

Gel Electrophoresis Characterization

C0051 and B0015

OprF

That was relatively successully extracted OprF from colonical PCR.

R0062

Preparation for Homologous Recombination: Plasimid Digestion

Enzyme from Thermo Scientific FastDigest Enzyme with 10X Green Buffer

Protocol as following:

• Nuclease-free water: 15ul
• pSB1C3: 1ul
• 10* FastDigest Green Buffer: 2ul
• EcoRI: 1ul
• SpeI: 1ul

Incubate solution at 37 degree celcius about 2h.

PCR of C0061,B0015 and micF

material:
C0061 plasmid: 477.1 ng/uL(diluted 20-folds)
B0015 PCR product:7.3 ng/uL
micF-1 PCR product:13.4 ng/uL

ForB0015 Primers produced from Sangon Biotech Co.,Ltd.

protocol:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl Primer-Fd
• 1μl Primer-Rv
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• 1uL C0061 plasmid,4uL B0015 plasmid,2uL plasmid
• (13.35-X)μl ddH2O

Renaturation at 55℃, extend for 1min

PCR of C0061,B0015,R0062,R0010 and C0062

material:
C0061 plasmid:477.1 ng/uL(0.2 uL)
B0015 plasmid:19 ng/uL(3 uL)
R0062 plasmid:143.3 ng/uL(0.5 uL)
C0051 plasmid:171.7 ng/uL(0.5 uL)
R0010 plasmid:357.7 ng/uL(diluted 10-folds,1 uL)
C0062 plasmid:739.4 ng/uL(diluted 10-folds,1uL)
for R0062.R0010 and C0062Primers produced from Sangon Biotech Co.,Ltd.

protocol:

• 2μl Taq buffer with KCl
• 0.4μl dNTPs
• 1μl Primer-Fd
• 1μl Primer-Rv
• 0.25μl Taq DNA Polymerase
• 2μl MgCl2
• XuL plasmid
• (13.35-X)μl ddH2O

Renaturation at 55℃, extend for 1min
PS:
C0061:656bp B0015:142bp R0062:104bp
C0051:803bp C0062:813bp R0010:243bp

Characterization of micF and SoxS by Gel Electrophoresis

micF were successfully PCRed.

Characterization of pSB1C3 and SoxS by Gel Electrophoresis

Characterization of R0051, C0061 and C0051 by Gel Electrophoresis

Plasimid Extraction for pSB1C3 and C0061

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

Gel Extraction

Material including: R0051, C0051 and C0061

Preparation:

1. Check out whether ethyl alcohol is added into Wash Solution.
2. Check out whether Buffer B2 has sediment.
3. Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

1. Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
2. Measure out the weight of tubes of gel.
3. Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
4. Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
5. After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
6. Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
7. Do the step 5 again.
8. Centrifuge the empty columns at 9000xg about 1min.
9. Open the columns and air them about 10 mins.
10. Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
11. Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

Characterization of B0015 and R0051 by Gel Electrophoresis

Overlap Circuit

This sensing circuit including promoter micF and SoxS.

Overlapping Fragments

In order to ligate the fragements micF or SoxS, C0061 and B0015, we need to make the equal mole proportion.

The mole of DNA molecule is calculated in the following ways:

among which, c is the concentration of DNA, V is the volumn required and n is the length of DNA. Because of the same molar mass of As, Gs, Cs and Ts, computation of same molar mass is omitted.

According to our sample extracted from PCR and calculation, ingradients characterize as below:

The theoratical volumn proportion is:

• SoxS-C0061-B0015: 5: 7: 3
• micF-C0061-B0015: 5.5: 7: 3

Firstly, to ligate these parts, we need 10 cycles without primer:
for circuit SoxS-C0061-B0015:

for circuit micF-C0061-B0015:

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 1min
4. 4 celcius degree for preserveation.

Then, we extracted 5 ul as template, and 15 ul adding with primer:
Primers were synthesized from Sangon Biotech. Co.,Ltd.

5ul-template protocol:

15ul-protocol:

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 1min
4. 4 celcius degree for preserveation.

PCR of Five Parts

material:
R0010 plasmid:193.1ng/uL
C0062 plasmid:952.5ng/uL
B0015 plasmid:93ng/uL
R0062 plasmid：143.3ng/uL

For R0051 Primers produced from Sangon Biotech Co.,Ltd.

protocol:

• 2uL 10X buffer with KCl
• 0.4uL dNTP
• 1uL primers-F
• 1uL primers-R
• 0.25uL Taq Polymerase
• 2uL MgCl2
• 1uL plasmid
• 12.35 uL ddH2O

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 1min
4. 4 celcius degree for preserveation.

Overlap PCR of bacteria III

material:

• R0062 PCR product:21.2 ng/uL
• C0051 PCR product:22.8 ng/uL
• B0015 PCR product:24.6 ng/uL

protocol:
total volume 20 uL

• R0062(104bp) 1 uL
• C0051(803bp) 8 uL
• B0015(153bp) 1.5uL
• ddH2O 4.85 uL
• buffer 2uL
• dNTP 0.4uL
• Taq polymerase 0.25uL
• MgCl2 2uL

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 1min
4. 4 celcius degree for preserveation.

After the 10 cycles,we divide the 20 ul system into two parts:5ul and 15ul.The 5ul PCR product will be used as template in another new 20ul system.On the other hand, add primers
(R0062-F primer 1uL;C0062-B0015-R(I) primer 1uL)to the remaining 15 uL PCR product.

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 1min
4. 4 celcius degree for preserveation.

Result:
we don't get the product we want.We guess the templates are not the right products,maybe they have mutations .So we decide to do the overlap PCR of bateriaI to test whether the method is suitable.

Plasmid Extraction of gRNA-AcrB and gRNA-EmrE

Procedure including:
1.Absorb 6 mL bacteria solution in to EP tubes, centrifuge them at 12,000xg 1mins and then discard the culture medium.
2.Add 250 ul Buffer S1 into sediment, and use spearhead to make bacteria suspended.
3.Add 250 ul Buffer S2, and overturn the EP tubes 8 times immediately and tenderly. Stand tubes about 3 mins.
4.Add 350 ul Buffer W3 and overturn the EP tubes 10 times again immediately and tenderly.
5.Centrifuge tubes at 12,000xg about 10 mins.
6.Pour the supernatant liquid into absorption columns ,centrifuge them at 12,000xg about 30 sec and then discard the liquid in the collecting pipe.
7.Add 500 ul Buffer W1 and centrifuge them at 12,000xg about 30 sec. Then discard the liquid in the collecting pipe.
8.Add 700 ul Buffer W2, centrifuge them at 12,000xg about 30 sec and discard the liquid in the collecting pipe.
9.Do the step 8 again.
10.Centrifuge the empty columns at 12,000xg about 1 min.
11.Put the columns in clean 1.5mL EP tubes, add 80 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 12,000xg.

Gel Extraction

Material including: R0061

Case 1: Sangon Biotech.

Preparation:

1. Check out whether ethyl alcohol is added into Wash Solution.
2. Check out whether Buffer B2 has sediment.
3. Adjust the thermostat water bath at 50 degree centigrade.

Procedure:

1. Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
2. Measure out the weight of tubes of gel.
3. Add Buffer B2 3 to 6 times the volume of gel and then incubate the EP tubes in thermostat water bath about 50 degree centigrade about 10 mins.
4. Optional: If the length of the DNA strip is shorter than 500 bp, it would be better add isopropanol one third volume of Buffer B2.
5. After gel is all dissolved, pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
6. Add 500 ul wash solution in a centrifuge at 9000Xg about 30s and pour out the solution.
7. Do the step 5 again.
8. Centrifuge the empty columns at 9000xg about 1min.
9. Open the columns and air them about 10 mins.
10. Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
11. Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

Case 2: Axygen

Procedure:

1. Cut up the effective bands under UV light and put the fragments into EP tubes with series numbers.
2. Measure out the weight of tubes of gel.
3. Add 400~600 ul Buffer DE-A and then incubate the EP tubes in thermostat water bath about 75 degree centigrade within 10 mins.
4. After gel is all dissolved, add 1/3 volumn of Buffer DE-B into EP tubes. The solution will turn yellow, illustrating complete dissolved.
5. Pour the solutions into absorption columns and centrifuge columns at 8000xg about 30s.
6. Add 500 ul Buffer W1 in a centrifuge at 9000Xg about 30s and pour out the solution.
7. Add 700 ul Buffer W2 in a centrifuge at 9000Xg about 30s and pour out the solution.
8. Centrifuge the empty columns at 9000xg about 1min.
9. Open the columns and air them about 10 mins.
10. Put the columns into clean 1.5 mL EP tubes and add 15-40uL Elution Buffer(2.5mM Tris-HCL,PH 8.5)(also can be substituted by TE or double distilled water). Stand about 1 min and then centrifuge at 9000xg about 1 min.
11. Keep the DNA solution and often measure the concentration of DNA and A260/A280 for further study.

Overlapping Fragments

We conduct the overlapping again.
However, the sample has a little difference. The concentration of micF changes to 58.7ng/uL. According to our sample extracted from PCR and calculation, ingradients characterize as below:

The theoratical volumn proportion is:

• SoxS-C0061-B0015: 5: 7: 3
• micF-C0061-B0015: 1.3: 7: 3

Firstly, to ligate these parts, we need 10 cycles without primer:
for circuit SoxS-C0061-B0015:

for circuit micF-C0061-B0015:

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 1min
4. 4 celcius degree for preserveation.

Primers are the same as before.

15ul-protocol:

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1min)
3. Elongation for whole at 72 degree celcius 1min
4. 4 celcius degree for preserveation.

PCR of Cas9(80l)

material:

8 uL buffer with KCl
35 uL dd water
8 uL Plasmid with Cas9
4 uL primer R
4 uL primer F
8 uL MgCl2
8 uL dNTPs
5 uL DMSO
1 uL Tap polymerase

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)
3. Elongation for whole at 72 degree celcius 1min
4. 4 celcius degree for preserveation.

Negative Chemotaxis Characterization

Done by Wang Luyao
Term 1 results

I put the paper on this plate after the E.coli grows homogeneously， so let's see the result later.

the controlled plate

the last plate was contaminated

Term 2

material:

LB solid medium plates x6

0.8、0.6、0.4、0.2、0.1g/ml Chloramphenicol

E.coli Top10

filter paper

Enzyme Digestion

Performing twice separately

Both as following protocol:

Overlap PCR of bacteriaI

material:

• micF PCR product :33.6ng/uL
• C0061 PCR product: 18.5ng/uL
• B0015 PCR product:9.2ng/uL
• micF-F primer
• B0015-R primer

protocol:
total volume: 25uL

• Taq Master Mix 12.5uL
• micF 2uL
• C0061 6uL
• B0015 2.6uL
• ddH2O 1.9uL

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 10 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 90s)
3. Elongation for whole at 72 degree celcius 10min
4. 4 celcius degree for preserveation.

After the 10 cycles,we divide the 20 ul system into two parts:5ul and 15ul.The 5ul PCR product will be used as template in another new 20ul system.On the other hand, add primers
to the remaining 15 uL PCR product.

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 90s)
3. Elongation for whole at 72 degree celcius 10min
4. 4 celcius degree for preserveation.

Results:
We don't get the product we want.We make some research and guess that the probable reasons are the following:Taq polymerase can cause 3'd A to PCR product;the annealing temperature is unsuitable;the length of overlap fragment isn't enough .

Negative Chemotaxis Characterization

Done by Wang Luyao
7.30

Term 2 results

The 0.8、0.6、0.4 plates just look similar, the denser Chloramphenicol the paper hold， the bigger the clear zone is.

but the 0.2 and 0.1g/ml plates are different, Top10 grows denser on the edge of the clear zone, I supposed it can show that E.coli has moved. So next time I'll try under 0.2g/ml.

the controlled plate

PCR of C0062 and R0010

material:

• 10 ul Taq PCR Master Mix
• 7 ul ddH2O
• 1 ul Primer F
• 1 ul Primer R
• 1 ul C0062 or R0010

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)
3. Elongation for whole at 72 degree celcius 1min
4. 4 celcius degree for preserveation.

Negative Chemotaxis Characterization

Done by Wang Luyao
Term 3, material:

• LB solid medium plates x5

• 0.2、0.1、0.05、0.025g/ml Chloramphenicol

• E.coli Top10

• filter paper

PCR of micF, SoxS, C0061 and R0015

material(volume 50uL):

• 25 ul Taq PCR Master Mix
• 15 ul ddH2O
• 2.5 ul Primer F
• 2.5 ul Primer R
• 2.5 ul Top10 culture, Top10 culture, C0061 or R0015

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 4.5 min)
3. Elongation for whole at 72 degree celcius 1min
4. 4 celcius degree for preserveation.

PCR of tRNA

Protocol (volume 20uL):

• 2 ul Taq Buffer
• 0.4 ul dNTP
• 1 ul tRNA-F
• 1 ul tRNA-R
• 0.25 ul Taq DNA Polymerase
• 2 ul MgCl2
• 1 ul tRNA
• 12.35 ul ddH2O

Protocol (volume 20uL):

• 2 ul Taq Buffer
• 0.4 ul dNTP
• 1 ul tRNA-F2
• 1 ul tRNA-R
• 0.25 ul Taq DNA Polymerase
• 2 ul MgCl2
• 1 ul tRNA
• 12.35 ul ddH2O

PCR cycle including:

1. Preheat: 94 degree celcius, 7min
2. 25 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s ) and elongation(72 degree celcius 1.5 min)
3. Elongation for whole at 72 degree celcius 10min
4. 4 celcius degree for preserveation.

Result
Only one sample get PCR product, concentration 441ng/μL. tRNA-F2 dosen't work for wrong template.
Remember to review gene map before experiments!

PCR of cheZ

material:
Top 10 Bacterial Solution

Primers produced from Sangon Biotech Co.,Ltd.

for cheZ (~600bp)
Protocol as this:

• 2.5 μl Taq buffer with KCl
• 2.5 μl dNTPs
• 0.75 μl cheZ-F Primer
• 0.75 μl cheZ-T7-R Primer
• 0.5 μl Kod DNA Polymerase(600bp/min)
• 1.0 μl MgCl2
• 2 uL Top10 Solution
• 15 μl ddH2O

PCR cycle including:

1. Preheat: 94 degree celcius, 5min
2. 35 cycles containing: degradation(94 degree celcius, 30s; annealing(55 degree celcius, 30s and elongation(72 degree celcius 1 min 30s)
3. Elongation for whole at 72 degree celcius 2min(C0061)or 1min(B0015 and R0062)
4. 4 celcius degree for preserveation.

It turned out we used wrong primer:(

PCR of CheZ and B0015

material:

• TOP 10 baterial solution
• PAO1 bacterial solution
• B0015 plasmid :93.0 ng/uL

protocol:
total system 25uL

• buffer 2.5 uL
• dNTPs 2.5uL
• MgSO4 2uL
• primer-F 0.75 uL
• primer-R 0.75 uL
• bacterial solution/plasmid 1uL
• KOD-Plus enzyme 0.5uL
• ddH2O 17uL

PCR cycle including:

1. Preheat: 94 degree celcius, 2min
2. 35 cycles containing: degradation(94 degree celcius, 15s; annealing(55 degree celcius, 30s and elongation(68 degree celcius 50s)
3. Elongation for whole at 68 degree celcius 7min.
4. 4 celcius degree for preserveation.

The protocol is from lab319,the enzyme is KOD-Plus.

Result:
We don't get the product we want .It turned out because of wrong primer we implemented.

Transformation of C0062

the protocol as following
(1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
(2)Absorb 1 uL or 2 uL plasmid and mix it with bacteria solution thoroughly.
(3)Put the tubes on the ice about 30 mins.
(4)Make a heat shock at 42 degree centigrade about 60 sec
(5)Put the tubes on the ice about 3 mins again.
(6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
(7)Centrifuge them at 4,000xg about 15 sec and we will se sediment in the tubes.
(8)Discard the supernatant liquid and leave about 100ul medium.
(9)Coat plate: add 100ul solution in a plate