iGEM 2017 — Team HBUT-China

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# Team:HBUT-China/Notebook

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# Notebook

* Molecular experiment log
* Microbiological experiment log

### Molecular experiment log

Experimental Objectives:

Construction of recombinant plasmids containing the pncrA promoter and the NcrB protein sequence And try to construct a recombinant plasmid containing the complete component.

![this is a photo](https://static.igem.org/mediawiki/2017/8/87/Notebook-1.png)

Figure 1 Plasmid map of bacteria

![this is a photo](https://static.igem.org/mediawiki/2017/a/a0/Notebook-2.png)

Figure 2 Bacteria of biological bricks

7.21-7.29

Topic: PCR amplification amplification pncrA and repressor protein NcrB and agar making

Materials:

2 × Buffer 100 μl

DD H2O 92 μl

pncrA forward primer 1 μl

pncrA reverse primer 1 μl

NcrB forward primer 1 μl

NcrB reverse primer 1 μl

The plasmid 4 μl

Agarose 0.3g

1 × TAE 30ml

1/10000 Bio Teke Dye 3 μl

2000bp maker4μl

10 × Buffer 8 μl

Procedure:

1. 5 μl of Buffer, 23 μl of DD H2O, 1 μl of plasmid, and 0.5 μl of forward primer and 0.5 μl reverse primer were mixed into the tube for a total of 50 μl. The mixture was divided and divided into two tubes and placed in a PCR instrument. PCR final system is the 25μl .There are 8 in total, of which 1, 2, 3, 4 marked N; 5,6,7,8 marked P.

PCR parameters set: denaturation 94 ° C, refolding 60 ° C, extended 72 ° C, 30 cycles.

2. Take the 0.3 g agarose microwave oven and heat it in 30 ml of 1 × TAE (prepare 1% agarose gel).

3. Add 3μl of dye to the hot mix and Pour into the mold .If bubbles are generated, removeing them with plastic pipettes.

4. Let the gel solidify for 30 min.

5. After the gelation, suit remove the comb. Place it in the gel tank.

6. At the end of the PCR, take out 8 tubes and 8 μl were mixed with 1 μl of 10 × Buffer and added to each well. At the end of PCR, 8 tubes were removed and then 8 μl were mixed with 1 μl of 10 × Buffer and added to each well. When the point of time, the tip of the gun is perpendicular to the filling hole so that the corner just under the opening in the sample hole. Add the sample and the mark maker slowly to the sample hole

7. Run the gel electrophoresis at 114V for 30 minutes.

8. Press the glue

Results and analysis:

![this is a photo](https://static.igem.org/mediawiki/2017/3/38/Notebook-3.jpg)

Fig 3 .PCR products of pncrA and NcrB in plasmid

Note: M: DL2000 DNA marker

The promoter (pncrA) and repressor (NcrB) were successfully amplified.

1. The first system settings: 50μl system, the primer plus 1μl

Results: There are many non-specific amplification

Analysis: Because the primer concentration is too much, there are many non-specific amplifications

2. The second system settings: 25μl system, the primer plus 0.5μl

Results: As shown in the figure, the nonspecific bands are less, the target bands had no obvious tailing and are clearly visible.

Analysis: The product was successfully amplified and the next step was purified

Topic:DNA recovery

Materials:

Agarose 0.6g

1×TAE 60ml

1/10000 Bio Teke Dye 6 μl

2000bp maker 8μl

10×Buffer 8 μl

Amplified fragment promoter (pncrA)

Amplified fragment repressor protein (NcrB)

DNA purification recovery kit (TIANGEN company)

Procedure:

Use DNA purification recovery kit and then run the glue to see the recovery effect

Results and analysis:

![this is a photo](https://static.igem.org/mediawiki/2017/0/0c/Notebook-4.jpg)

Fig4. Pecturing of pncrA and NcrB

![this is a photo](https://static.igem.org/mediawiki/2017/0/0f/Notebook-5.png)

Fig5. Recovery of pncrA and NcrB

Glue recycling are successful and no other specific bands.

Topic: Digestion

Materials:

Plasmid vector PSB1C3 10 μl

Promoter (pncrA) 10 μl

Repressor protein (NcrB) 10 μl

restriction enzyme (Ecor1) 1 μl

restriction enzyme (Pst1) 1 μl

10 × Buffer O 2μ

Agarose 0.6 g

1×TAE 60ml

1/10000 Bio Teke Dye 6 μl

2000bp maker 8μl

10 × Buffer 3 μl

DNA purification recovery kit (TIANGEN company)

Procedure:

Digestion System:1 μl of Ecor1, 1 μl of Pst1, 10 μl of digested DNA, 10 × Buffer O 2 μ, 6 μl of distilled water, total 20μl.

Configure the mixture according to the system

37 ° C for 2 hours

65 ℃ fire for 20 minutes

Purification of the digestion product using a DNA purification recovery kit and running gel.

Results and analysis:

Digestion effect is not too good and the band is not bright enough.

Probably because the concentration of the target fragment we added was insufficient.

![this is a photo](https://static.igem.org/mediawiki/2017/c/c9/Notebook-6.png)

Fig7. Digestion of Pncra and Ncrb

None. Results are expected after transformation and PCR.

7.29-8.4

Topic:Ligation

Materials:

Digested PSB1C3 2μl

Digested NcrB 5μl

Digested pncrA 8μl

T4 DNA ligase 1μl

10xT4 Buffer 2μl

DD H2O 19μl

Procedure:

Connection system: The total volume is 20μl

Linear plasmid vector: 1μl，about 30 ng (measured plasmid backbone concentration of 29.2 ng /μl)

Inserted DNA:plasmid backbone = 1: 1-5: 1 ，and we select ratio of 3: 1

Digested NcrB: 5μl (measured at a concentration of 16.7 ng /μl)

Or digested pncrA: 8μl (measured at a concentration of 11 ng /μl)

10xT4 Buffer: 2μl

T4 DNA ligase: 1μl (Thermo Corporation, No. EL0014, enzyme activity 5 weiss U /μl)

added DD H2O to 20 μl

Configure the mixture according to the system above

22 °C for 40 minutes

65 °C inactivated for 20 minutes

Results and analysis:

None. Results are expected after transformation and PCR.

Topic: Transformation

Materials:

Recombinant plasmid of ncrB DNA 2 μl

Recombinant plasmid of pncrA DNA 2 μl

DH5α bacterium 200 μl

SOC 800 μl

Chloramphenicol 200 μl

Agar Petri dish 8

Procedure:

1. Take a commercial competent cells (total 100μl), two self-made competent cells (each 80μl), put frozen on ice 15min

2. Prepare four sterilized 2mlEP tubes, put on ice to precool (This should be carried out at the same time with the first step )

3. Draw 50 μl of competent cell fluid added to the four precooled EP tube (two commercial, two self-made)

4. Add 1μl recombinant plasmid to each of the commercial competent cells and self-made competent cells. Do the same to the pncrA DNA.

5. Close the EP tube, and then put it in an ice bath for 30min

6. 42°C heat shock for 1min, put it in an ice bath for 5min

7. Add 200μl of SOC medium to each EP tube

8. 37 °C shaker culture 3h

9. Take two samples, one 50 μl and one 150 μl, of each EP tube, place onto the plate and label them.

10. place in incubator at 37C for 12 to 16 hours.

Results and analysis:

![this is a photo](https://static.igem.org/mediawiki/2017/c/c8/Notebook-7.png)

Fig 7.Transformation of pncrA

![this is a photo](https://static.igem.org/mediawiki/2017/5/57/Notebook-8.png)

Fig8.Transformation of NcrB

The recombinant plasmid with pncrA didn’t transform into E. coli, the recombinant plasmid with ncrB transformed into E. coli successfully. The DNA gel electrophoresis results showed digestion succeeded, thus there was no conoly due to the failure of ligation and transformation.

Topic: PCR of pncrA

Materials:

2 × taq enzyme 75 μl

pncrA forward primer 1.5 l

pncrA reverse primer 1.5 μl

DD H2O 72μl

Agarose 0.3g

1 × TAE 30ml

1/10000 Bio Teke Dye 3 μl

2000bp maker 4μl

10 × Buffer 6 μl

bacteria

Procedure:

PCR MIX system: the total volume is 25μl. 2×taq enzyme 25μl ( from Engine company), 0.5μl forward and 0.5μl reverse primer, DDH2O 24μl, the total is 50μl. Then divide them into two tubes. Repeat the operations for other two times, and there are 6 tubes totally.

A single colony was randomly selected at room temperature, and was picked with a sterile toothpick or a tip, and a copy was made on a solid plate and added to 500μl of liquid LB medium. The colonies were added to the PCR MIX system in the same manner. Do a colony PCR to verify the part size.

Results and analysis:

![this is a photo](https://static.igem.org/mediawiki/2017/7/7f/Notebook-9.png)

Fig9. PCR of Pncra

There was a successful connection of pncrA DNA bands, which may have a small amount of specific amplification. the recombinant plasmid with ncrB transformed into E. coli successfully, the problem may be either ligation or Transformation, but ligation is more likely.

8.4-8.11

Topic:Ligation

Materials:

Digested PSB1C3 4μl

Digested NcrB 6μl

Digested pncrA 6μl

T4 DNA ligase 2μl

10xT4 Buffer 2μl

Procedure:

Connection system: The total volume is 10μl.

Linear plasmid vector: 2μl

Interested DNA: plasmid backbone = 1: 1-5: 1 ，and we select ratio of 3: 1

Digested Ncrb: 6μl

Or digested Pncra: 6μl

10xT4 Buffer: 1μl

T4 DNA ligase: 1μl

added DD H2O to 20 μl

Configure the mixture according to the system

22 °C for 4 hours

65 ℃ inactivated for 30 minutes

Results and analysis:

None. Results are expected after transformation and PCR.

Topic:Transformation

Materials:

ligated plasmid of ncrB DNA 2 μl

ligated plasmid of pncrA DNA 2 μl

DH5α bacterium 100 μl

Chloramphenicol 200 μl

LB medium 1 mL

LB solid medium 50 mL

Chloramphenicol 50 μl

Agar Petri dish 2

Procedure:

1. Take two self-made competent cells (each 80μl) and put on ice to freeze - 15min

2. Prepare two sterilized 2ml EP tubes, put on ice to precool (This should be carried out at the same time as the first step )

3. Draw 50 μl of competent cell fluid and add to the two precooled EP tubes

4. Add 1μl recombinant plasmid to each of the commercial competent cells and self-made competent cells. Do the same to the pncrA DNA.

5. Close the EP tube, and then put it in an ice bath for 30min

6. 42°C heat shock for 90s, put it in an ice bath for 5min

7. Add 200 μl of SOC medium to each EP tube

8. 37 ℃ shaker culture 3h

9. Take 50 μl and 150 μl of each EP tube onto the plate and label them

10. 37 ° C incubating for 12 to 16 hours

Results and analysis:

![this is a photo](https://static.igem.org/mediawiki/2017/6/6a/Notebook-10.png)

Fig 10. Transformation of pncrA and ncrB

DH5α were successfully transformed and the colonies were grown on the medium.

Topic:PCR of pncrA and ncrB

Materials:

2xtaq enzyme 125μl

pncrA forward primer 2.5μl

pncrA reverse primer 2.5μl

ncrB forward primer 2.5μl

ncrB reverse primer 2.5μl

DD H2O 120μl

bacteria

Agarose 0.6g

1 × TAE 60ml

1/10000 Bio Teke Dye 6 μl

2000bp maker4μl

10 × Buffer 12 μl

Procedure:

PCR MIX system: the total volume is 25μl.

2×taq enzyme 25μl (from Engine company),

0.5μl forward and reverse primer,

DD H2O 24μl, the total is 50μl.

Then divide them into two tubes. Repeat the operations for other 5 times, and there are 10 tubes totally.

A single colony was randomly selected at room temperature, and was picked with a sterile toothpick or a tip, and a copy was made on a solid plate and added to 500μl of liquid LB medium. The colonies were added to the PCR MIX system in the same manner. Do a colony PCR to verify the part size.

Results and analysis:

![this is a photo](https://static.igem.org/mediawiki/2017/4/4a/Notebook-11.png)

Fig11. PCR of pncrA and ncrB

We improve the ligation system, extend the ligation time, optimize the transformation step. We try to make ncrB DNA, pncrA and plasmid vector connected and its successful transformation.

Topic:sequencing

Procedure:

We will send the samples to company for further sequencing.

Results and analysis:

![this is a photo](https://static.igem.org/mediawiki/2017/f/f9/Notebook-12.png)

Fig12.Comparison of ncrB

![this is a photo](https://static.igem.org/mediawiki/2017/e/ea/Notebook-13.jpg)

Fig13.Comparison of pncrA

After sequencing, we confirmed that the DNA is indeed what we need.

8.12-8.23

Topic: Digestion，Ligation，Ligation

Procedure:

The method of digestion and ligation is the same as above.

Transformation: The system and method are the same as before, except that the colonies are not required to be coated with a solid culture medium and can be directly cultured on a liquid medium overnight to extract the plasmid. The transformed plasmids are E1010, B0010, B0012, B0032, B0034, J23102.

Results and analysis:

B0010 failed to convert and there were no Colonies, but other plasmids successfully transformed.

Topic:3A assembly

Materials:

Linear plasmid vector (PSB1T3) 10μl

J23102 10μl

B0032 10μl

B0034 10μl

Digested of pncrA 5μl

T4 DNA ligase 4μl

10xT4 Buffer 3μl

restriction enzyme (EcoRI) 4μl

restriction enzyme (Pst1) 4μl

Restriction enzyme (XbaI) 2μl

restriction enzyme (SpeI) 2μl

10 × Buffer O 12μl

10 × Buffer T 6μl

DD H2O 18μl

Procedure:

The method of digestion, ligation and transformation is the same as above. And then use the mention the plasmid kit.

![this is a photo](https://static.igem.org/mediawiki/2017/b/bb/Notebook-14.png)

Fig14. Principle of 3A assembly

The clips are joined in this order:

J23102-B0032-ncrB-B0010-B0012-pncrA-B0034-E1010-B0012-B0034.

We first use the 3A method toconnect thesefragments:

J23102-B0032-ncrB; B0010-B0012-Pncra-B0034; E1010-B0012-B0034.

The "three large fragments" were then ligated using Gibson assembly technology (seamless splicing), and finally transformed into the PSB1C3 plasmid.

Results and analysis:

Because of limited time, we regret that we have not been able to complete all the experiments. but we have successfully confirmed the J23102-B0032 connection to the feasibility of our idea.

### Microbiological experiment log

2017/07/05

Topic: Bacteria Culturing

Material:

LB 50mL

chloramphenicol

E.coli(K2304002)

5x test tube

Procedure:

1. Sterilize test tubes and LB media.

2. Distribute the culture medium and chloramphenicol into the test tube. Each tube contained 5 ml of LB medium containing 5 μL of chloramphenicol.

3. Inoculate 1% of E.coli(K2304002) in the LB medium.

4. Culture the samples at 200rpm, 37 ℃overnight.

2017/07/06

Topic: Test K2304002

Material:

E.coli(K2304002) cultured on July 5

Nickel ion solution (0.5mol/L)

Procedure:

1. Add the nickel ion solution to the bacteria solution.

2. Continue to culture bacteria for 2 hours.

3. Observe whether the bacterial solution changes.

4. Centrifuge the bacteria at 12,000ram for 10 minutes.

5. Observe the solution after centrifugation.

Result:

After 2 hours of culture, the bacteria solution did not change. We are very worried that K2304002 does not work.

![this is a photo](http://123.207.175.242/csdn/img/note/hbut-new-note1.png)

But after centrifugation, The red precipitate appeared at the bottom of the centrifuge tube，while the blank control group produced very little, or none of the red precipitation. This indicates that KL2304002 can be induced by Ni ions to produce RFP.

![this is a photo](http://123.207.175.242/csdn/img/note/hbut-new-note2.png)

2017/07/17

Topic: Nickel ion tolerance

Material:

LB 200mL

chloramphenicol

E.coli(K2304002)

30x test tube

ddH2O

Nickel ion solution (5x102mol/L、5x101mol/L、 5x100mol/L、5x10-1mol/L、5x10-2mol/L、5x10-3mol/L、5x10-4mol/L)

Procedure:

1. Sterilize test tubes and LB media.

2. Distribute the culture medium and chloramphenicol into the test tube. Each tube contained 5 ml of LB medium containing 5 μL of chloramphenicol.

3. Each group of three tubes was added with the same concentration of nickel ions. One of test tube is blank, a total of eight test tubes.

4. Inoculate 1% of E.coli(K2304002) in the LB medium.

5. Culture the samples at 200rpm, 37 ℃overnight.

6. Measure the OD600 and FI of each sample.

Result:

![this is a photo](http://123.207.175.242/csdn/img/note/hbut-new-note3.png)

By analyzing the OD value, we draw the relationship between cell growth and nickel ion concentration.

![this is a photo](http://123.207.175.242/csdn/img/note/hbut-new-note4.png)

From the figure we can see that when the nickel ion concentration greater than 0.5mmol / L, the cell growth is inhibited.

2017/07/28

Topic: Growth curve

Material:

LB 1000mL

chloramphenicol

E.coli(K2304002)

8x beakerflask

Nickel ion solution (5x10-2mol/L、1x10-2mol/L、5x10-3mol/L、1x10-3mol/L、5x10-4mol/L

)

Procedure:

1. Sterilize beakerflask and LB media.

2. Distribute the culture medium and chloramphenicol into the beakerflask. Each beakerflask contained 100 ml of LB medium containing 100 μL of chloramphenicol.

3. Each beakerflask was added with different concentration of nickel ions. One of beakerflask is blank, a total of eight beakerflask.

4. Inoculate 1% of E.coli(K2304002) in the LB medium.

5. Culture the samples at 200rpm, 37 ℃.

6. Detection of OD and fluorescence intensity every other hour.

Result:

We find that the OD was close, but the fluorescence intensity was very different.

![this is a photo](http://123.207.175.242/csdn/img/note/hbut-new-note5.png)

he graph below shows that there is a correlation between fluorescence intensity and nickel ion concentration.

![this is a photo](http://123.207.175.242/csdn/img/note/hbut-new-note6.png)

After this experiment, we further measured the 24-hour growth cuve.

![this is a photo](http://123.207.175.242/csdn/img/note/hbut-new-note7.png)

2017/08/10

Topic: Specificity

Material:

LB 100mL

chloramphenicol

E.coli(K2304002)

5x test tube

Cd2 +, Co2 +, Cu2 +, Fe3 +, and Ni2 +solution

Procedure:

1. Sterilize test tubes and LB media.

2. Distribute the culture medium and chloramphenicol into the test tube. Each tube contained 5 ml of LB medium containing 5 μL of chloramphenicol.

3. Each test book was added with different ions. One of test tube is blank, a total of six test tube.

4. Inoculate 1% of E.coli(K2304002) in the LB medium.

5. Culture the samples at 200rpm, 37 ℃ overnight.

6. Observe them with a fluorescence microscope.

Result:

Clearly, only the sample with Ni ions showed red fluorescence, the other groups had no red fluorescence. Note that the iron ions reacted with the substance in the culture medium，so that there is an impurity in the field of view.

![this is a photo](http://123.207.175.242/csdn/img/note/hbut-new-note8.png)

2017/08/15

Topic: Qualitative testing

Material:

LB 100mL

chloramphenicol

E.coli(K2304002)

9x test tube

Nickel ion solution (1x10-5mol/L、5x10-6mol/L、1x10-6mol/L)

Procedure:

1. Sterilize test tubes and LB media.

2. Dilute the cultures to a target OD of 0.8.

3. Each test tube was added with different concentration of nickel ions.

4. Inoculate 1% of E.coli(K2304002) in the LB medium.

5. Culture the samples at 200rpm, 37 ℃ 2 hours.

6. Detection of OD and fluorescence intensity every other hour.

Result:

We found that this method can be uesd to find the linear relationship between nickel ions and fluorescence intensity.

![this is a photo](http://123.207.175.242/csdn/img/note/hbut-new-note9.png)

This graph shows that after 2 hours, the nickle ion concentration is positively correlated with the fluorescence intensity. Continue to experiment, we can find a suitable means of detecting nickle ion.

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