iGEM 2019 — Team SEFLS_Shanghai

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# Team:SEFLS Shanghai/Notebook

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![](https://static.igem.org/mediawiki/2019/a/a9/T--SEFLS_Shanghai--backgroundIMG.jpg)

Notebook

Notebook

#### Our Original Notes

You can view below our original notes in Chinese.

# Project：Squalene production by E.coli

# Team：SEFLS

# Date：2019-4-18 to 2019-10-12

# Day1

按照真菌基因组DNA提取试剂盒提取解脂耶氏酵母和酿酒酵母的基因组；

按照细菌基因组DNA提取试剂盒提取枯草芽孢杆菌的基因组；

对提取的基因组进行琼脂糖凝胶电泳。

![](https://static.igem.org/mediawiki/2019/3/3f/T--SEFLS_Shanghai--notebook-p1.png)

M：λ-HindIII digest；1：解脂耶氏酵母基因组；2：酿酒酵母基因组。

# Day2

以提取的基因组为模板，使用高保真DNA聚合酶对鲨烯合酶YSS、NSS和KSS基因进行PCR；

以基因合成公司送来的含有基因thSQS和crtN的质粒为模板使用高保真DNA聚合酶对基因thSQS和crtN进行PCR；

![](https://static.igem.org/mediawiki/2019/a/ae/T--SEFLS_Shanghai--notebook-p2.png)

对PCR产物进行琼脂糖凝胶电泳，验证PCR条带正确后进行DNA凝胶回收；

对DNA凝胶纯化产物进行PCR鉴定；

![](https://static.igem.org/mediawiki/2019/b/bf/T--SEFLS_Shanghai--notebook-p3.png)

3.YSS; 4.NSS(酿酒酵母); 5.thSQS; 6.KSS(枯草芽孢杆菌)

pETDuet-1质粒进行100μL体系双酶切，37度过夜。

酶切体系

pETDuet-1

30 μL

pETDuet-1

30 μL

10×K

5 μL

10×K

10 μL

BSA(0.1%)

10 μL

none

none

BamHⅠ

5 μL

BamHⅠ

5 μL

NotⅠ

5 μL

HindⅢ

5 μL

ddH2O

45 μL

ddH2O

50 μL

**Day3**

对pETDuet-1酶切产物进行DNA凝胶回收；

测定pETDuet-1酶切产物和YSS、NSS、KSS、thSQS的DNA凝胶纯化产物的浓度；

按照克隆试剂盒的说明，根据测得的浓度值进行计算，最终确定连接反应体系，进行连接反应；

将连接反应产物对DH5α感受态进行转化。

![](https://static.igem.org/mediawiki/2019/0/02/T--SEFLS_Shanghai--notebook-p4.png)

连接反应体系

测定浓度(ng/μL)

最适用量

(ng)

实际添加量

(μL)

pETDuet-1（BamHⅠ/ NotⅠ）

13.1

108.4

8

pETDuet-1 (BamHⅠ/ HindⅢ）

12.8

108.4

8

KSS

60.3

40

1

NSS

130.1

64

0.5

thSQS

109.1

40

0.5

YSS

69.9

60

1

# Day4

挑单克隆培养5h后，对基因YSS、NSS、KSS和thSQS进行菌液PCR验证；

对菌液PCR产物进行电泳鉴定，对鉴定正确的阳性转化子进行保种，并取200微升菌液送去测序；

![](https://static.igem.org/mediawiki/2019/2/2c/T--SEFLS_Shanghai--notebook-p5.png)

# Day6

在液体LB培养基中转接BL21（DE3），为明天制备BL21（DE3）感受态做准备。

# Day7

对BL21（DE3）进行感受态制备；

将之前送去测序正确的转化子转接试管，液体LB培养基37度培养过夜；

# Day8

进行质粒转化实验，将质粒p35151转入BL21（DE3）感受态；

提取质粒，进行37度过夜双酶切， 为明天连接基因crtN做准备。

![](https://static.igem.org/mediawiki/2019/4/43/T--SEFLS_Shanghai--notebook-p6.png)

# Day9

对酶切产物进行DNA凝胶回收；

测定酶切产物和基因crtN的凝胶纯化产物浓度；

按照克隆试剂盒的说明，根据测得的浓度值进行计算，最终确定连接反应体系，进行连接反应，将反应产物保存在-20度；

挑取转化质粒p35151 的BL21（DE3）单克隆，37度过夜培养。

![](https://static.igem.org/mediawiki/2019/d/d7/T--SEFLS_Shanghai--notebook-p7.png)

连接反应体系

测定浓度(ng/μL)

最适用量

(ng)

实际添加量

(μL)

crtN

112.2

60

0.5

pET-YN

3.5

140

10

pET-NN

4.9

140

10

pET-KN

12.7

130

10

pET-thN

11.7

130

10

# Day10

对含有质粒p35151 的BL21（DE3）进行感受态制备；

将昨天的连接反应产物对DH5α感受态进行转化。

# Day11

挑单克隆培养5h后，对基因thSQS、KSS、NSS、YSS和crtN进行菌液PCR验证；

对菌液PCR产物进行电泳鉴定，对鉴定正确的阳性转化子进行保种，并取200微升菌液送去测序；

![](https://static.igem.org/mediawiki/2019/0/02/T--SEFLS_Shanghai--notebook-p8.png)

# D14

将之前送去测序正确的转化子转接试管，液体LB培养基37度培养过夜；

# D15

提取含有不同鲨烯合酶基因和crtN基因的质粒pET-KN、pET-NN、pET-YN和pET-thN；

将提取的以上质粒分别进行转化实验，质粒转入含有p35151 的BL21（DE3）感受态。

![](https://static.igem.org/mediawiki/2019/e/e9/T--SEFLS_Shanghai--notebook-p9.png)

# D16

每个转化平板上挑3个克隆37℃，250r/min过夜培养；

# D17

过夜培养的菌液按10%接种量转入含有3ml TB培养基的试管中，37℃，250 r/min培养至OD600为0.6-0.9，添加终浓度为0.1mM 的IPTG诱导，30℃，180 r/min培养48h。

# D18

对菌株N1、N2、N3和N4进行类胡萝卜素提取及检测；

对检测结果进行分析，结果显示鲨烯合酶YSS活性最高；

N1

N2

N3

N4（YSS）

0.003

0.741

0.749

1.175

0.004

0.829

0.959

1.226

0.003

0.522

0.706

1.085

将菌株N4转接试管，37℃培养过夜。

# D19

对菌株N4进行30℃和37℃发酵培养

过夜培养的菌液按10%接种量转入含有3ml TB培养基的试管中，37℃，250 r/min培养至OD600为0.6-0.9，添加终浓度为0.1mM 的IPTG诱导，在30℃和37℃两种温度下，180 r/min培养48h。

# D21

对在30℃和37℃两种温度下培养的菌株N4进行类胡萝卜素提取及检测；

对检测结果进行分析，结果显示The pigmentation level was very low at 37℃, 因此我们选择30℃为菌株的鲨烯合成发酵温度。

30℃

37℃

1.259

0.036

1.184

0.006

1.155

0.030

# D22

将pETDuet-1和pET-YSS分别转化含有 p35151的BL21(DE3)感受态。

# D23

转化平板上挑3个克隆转接试管，37℃，250r/min过夜培养；

# D24

过夜培养的菌液按10%接种量转入含有20ml TB培养基的摇瓶，37℃，250 r/min培养至OD600为0.6-0.9，添加终浓度为0.1mM 的IPTG诱导，30℃，180 r/min培养96h。

# D28

将摇瓶发酵96h后的菌液离心收集，进行鲨烯的提取与HPLC检测

HPLC检测结果：

样品

峰面积

鲨烯产量（mg/L）

1、标品

8688.8

160

2、阴性对照

0.0

0

3、样品1

1179.5

21.7

4、样品2

811.8

14.9

5、样品3

1098.5

20.2

菌株H1的鲨烯产量为 18.9 mg/L

# Day29

质粒pET-IAY、pET-IAY-CN、pET-HIAY和pMEP-DG的构建

1、获得目的片段YSS、dxs、ispH、ispG和IIA(rbs-idi-rbs-ispA)

基因dxs、ispH和ispG均从大肠杆菌中PCR获得，IIA片段以质粒p35151为模板PCR获得。

2、分别对骨架载体pBBR1MCS-2、pETDuet-1和pET-YN进行双酶切，pBBR1MCS-2是构建pMEP-DG的骨架，pETDuet-1是构建pET-IAY和pET-HIAY的骨架，而pET-YN是构建pET-IAY-CN的骨架；

3、将酶切回收的质粒骨架和凝胶回收目的片段进行电泳验证

4、将同源重组的片段与对应的骨架载体进行连接反应，反应体系组合如下：

pET-IAY：pET Duet-1酶切产物+IIA+YSS

pET-HIAY： pET Duet-1酶切产物+ispH+IIA+YSS

pET-IAY-CN：pET-YN酶切产物+ IIA+YSS

pMEP-DG：pBBR1MCS-2酶切产物+dxs+ispG

在转化过程中，我们遇到了一些困难，比如平板上长有很多克隆，但是我们获取不到阳性转化子。后来我们通过在PCR产物中添加DpnI酶解决了这个问题，推测可能在准备PCR反应液时，添加的质粒模板浓度过高，从而导致最后平板上长出了过多的阴性转化子。我们还遇到了平板上不长克隆的情况，后来通过提高质粒酶切产物的浓度和稀释片段回收产物的浓度得到解决，推测可能是由于质粒浓度过低或者片段浓度过高都可能影响连接反应体系的准确性，如果添加的质粒和连接的片段不在合适的范围内都有可能导致连接反应的失败。

5、挑取转化平板上的单克隆进行菌液PCR验证，验证结果说明我们成功构建了质粒pET-IAY、pET-IAY-CN、pET-HIAY和pMEP-DG。

# Day 30

质粒pMVA1和pMVA2的构建

1、 获得目的片段MVA1（rbs-AtoB-rbs-HMGS-rbs-tHMGR）和MVA2（rbs-MK-rbs-PMK-rbs-PMD-rbs-idi），以p35151为模板。

2、 酶切质粒骨架pBBR1MCS-1-(p15)和pBBR1MCS-2

3、 连接转化

4、 挑单克隆进行菌液PCR验证

# Day 31

质粒pUC-IAY和pUC-HIAY构建

1、 获得目的片段IAY（rbs-idi-rbs-ispA-rbs-YSS）和MVA2（rbs-ispH-rbs-idi-rbs-ispA-rbs-YSS），以pET-IAY和pET-HIAY为模板

2、 酶切质粒骨架pUC19；

3、 连接转化

4、 挑单克隆进行菌液PCR验证

# Day 32

鲨烯的发酵

对构建的菌株进行96h发酵，发酵数据如下：

菌株H1（mg/L）：21.7 14.9 20.2

菌株H2（mg/L）：63.3 78.9 65.6

菌株H3（mg/L）：64.3 47.4 39.5

菌株H4（mg/L）：326.6 250.7 317.5

菌株H5（mg/L）：537.6 378.2 501.1

菌株H6（mg/L）：650.5 577.9 625.1

菌株H7（mg/L）：921.8 1041.3 959.8

菌株XH3（mg/L）：293.9 280.6 306.3

菌株XH4（mg/L）：395.2 636.76 480.1

菌株XH5（mg/L）：1238.5 1297.2 1287.1


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# Team:SEFLS Shanghai/Experiments

* [Judging Form](https://igem.org/2019_Judging_Form?team=SEFLS_Shanghai)
* Awards

  [Model](https://2019.igem.org/Team%3ASEFLS_Shanghai/Model)
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3ASEFLS_Shanghai/Human_Practices)
  [Education & Engagement](https://2019.igem.org/Team%3ASEFLS_Shanghai/Public_Engagement)
* [Safety](https://2019.igem.org/Team%3ASEFLS_Shanghai/Safety)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3ASEFLS_Shanghai/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ASEFLS_Shanghai/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ASEFLS_Shanghai/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3ASEFLS_Shanghai/Part_Collection)
* Project

  [Description](https://2019.igem.org/Team%3ASEFLS_Shanghai/Description)
  [Design](https://2019.igem.org/Team%3ASEFLS_Shanghai/Design)
  [Experiments](https://2019.igem.org/Team%3ASEFLS_Shanghai/Experiments)
  [Notebook](https://2019.igem.org/Team%3ASEFLS_Shanghai/Notebook)
  [Contribution](https://2019.igem.org/Team%3ASEFLS_Shanghai/Contribution)
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  [Improve](https://2019.igem.org/Team%3ASEFLS_Shanghai/Improve)
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* Team

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* [71PERCENT](https://2019.igem.org/Team%3ASEFLS_Shanghai)
* ABOUT

![](https://static.igem.org/mediawiki/2019/a/a9/T--SEFLS_Shanghai--backgroundIMG.jpg)

* [Genome Extraction](#extraction)
* [PCR](#pcr)
* [Electrophoresis](#electrophoresis)
* [DNA Purification](#purification)
* [Plasmid Extraction](#plasmid-extraction)
* [Enzymatic system](#system)
* [Fragment Recycling](#recycling)
* [Connecting Plasmids](#connecting)
* [Cultivation](#cultivation)
* [Cell Preparation](#preparation)
* [Transformation](#transformation)
* [Colony Identification](#colony-identification)
* [Carotenoids Testing](#carotenoids)
* [Fermentation](#fermentation)
* [Squalene Extraction](#testification)
* [Chromatography](#analysis)

Experiments

Experiments

#### 1. Genome Extraction

The extraction process is carried out according to the instructions on the manual of the genome extraction reagent produced by Beijing Solarbio Science & Technology Co., Ltd.

1. Sample processing: Regarding the yeast, we take 1-2 mL of liquid containing cultured fungi and collect it using a centrifuge. The upper layer, which is clear liquid, is discarded. 200μl of solution A is added, followed by the adding of 20μl RNase A, and 100mg of glass bead. The new solution is oscillated with a high-speed oscillator for 5-10 minutes.
2. Add 20μl of proteinase K (10mg/ml), and shake it till the solution becomes stable. Put it in 55℃ water bath for 30 minutes, during which time we can reverse the centrifuge tube a few times. Put it in the centrifuge and run the centrifuge at 12000 rpm for 2 minutes and transfer the upper clear layer to a new centrifuge tube. This process should be repeated if there is any visible precipitate.
3. Add 200μl of solution B into the upper layer liquid, and shake until the solution stabilizes. If white precipitate is present in the solution, we can put it in 55℃ water bath for 5 minutes and the precipitate would disappear, and this will not affect our later experiment. If the solution had not turned clear, it would mean that the sample is not completely digested, which might result in the reduced purity or quantity of the extracted DNA and might also lead to blocking the adsorption column when it has been inserted，which will also prolong the digestion time.
4. Add 200μl of anhydrous ethanol and shake until the solution is stable. Floc may appear at this time, which will not affect the extraction of DNA. We then add everything in an adsorption column and wait for two minutes.
5. Put the solution in the centrifuge for 1 minute at 12000rpm and dispose of waste liquid. Add an adsorption column into the collection tube.
6. Add 600μl of rinse into the adsorption column and centrifuge the solution at 12000rpm for 1 minute, dispose of waste liquid and put the adsorption column in to the collection tube.
7. Add 600μl of rinse into the adsorption column and centrifuge the solution at 12000rpm for 1 minute, dispose of waste liquid and put the adsorption column in to the collection tube.
8. Centrifuge the solution at 12000rpm for 2 minute, and keep the adsorption column at room temperature or in a temperature-controlled environment(50℃) for a few minutes, the aim of which is to remove the remaining rinse in the adsorption column.
9. Put the adsorption column in a clean centrifuge tube and add in mid-air in the center of a adsorption film 50-200μl of spent reagent that has been kept in 65℃ water bath beforehand. Place it in room temperature for 5 minutes before centrifuging it at 12000rpm for 1 minute.
10. Add the spent reagent into the adsorption column again and keep it in room temperature for two minutes. Centrifuge it at 12000rpm for 2 minutes and we get the genome DNA in high quality.

#### 2. PCR

# Random PCR

Prepare the PCR mixture (on ice, mix very well)

|  |  |
| --- | --- |
| **Primers** (10μM) | 1 rxn (μl) |
| Primers-Fw | 2 |
| Primers-Rev | 2 |
| **PCR solutions** | 1 rxn (μl) |
| PrimeSTAR MAX Premix (2×) | 25 |
| Template | X |
| ddH2O | to 50 |

**Primers (Fw/Rev): 200μl Concentration: 10μM**

Dissolve to a concentration of 100μM.

Then dilute primer-Fw/Rev to 10μM.

Primermix:

20μl primer-Fw/Rev + ddH2O 180μl

**PCR-cycles**

|  |  |  |
| --- | --- | --- |
|  | **Temp (****℃****)** | **Time** |
| **Predenaturation** | 98 | 10 sec. |
| **3-step cycles 30 cycles** | | |
| a. Denaturation | 98 | 10 sec. |
| b. Annealing | Tm-5 | 10 sec. |
| c. Extention | 72 | 5 sec./kb |
| **Final extention** | 72 | 10 min |
| **Hold** | 10 |  |

The Annealing Temperature is set according to the characteristics of the primer sequence, while the prolonged time is set based on enzyme efficiency and the length of the amplified fragment.

**Colony PCR**

Prepare the PCR mixture (on ice, mix very well)

|  |  |
| --- | --- |
| **Primers** (10μM) | 1 rxn (μl) |
| Primers-Fw | 1 |
| Primers-Rev | 1 |
| **PCR solutions** | 1 rxn (μl) |
| UniTaq Master Mix (2×) | 10 |
| Template | X |
| ddH2O | to 20 |

**PCR-cycles**

|  |  |  |
| --- | --- | --- |
|  | **Temp (****℃****)** | **Time** |
| **Predenaturation** | 94 | 5min |
| **3-step cycles 30 cycles** | | |
| d. Denaturation | 94 | 30 sec. |
| e. Annealing | Tm-5 | 30 sec. |
| f. Extention | 72 | 30 sec./kb |
| **Final extention** | 72 | 5 min |
| **Hold** | 10 |  |

The Annealing Temperature is set according to the characteristics of the primer sequence, while the prolonged time is set based on enzyme efficiency and the length of the amplified fragment.

**Please see 4. PCR**

#### 3. Agarose Gel Electrophoresis on PCR Product

1. Determine the concentration of agarose based on the length of the DNA segment. The concentration used in this experiment are all 1.0%. Add 1% agarose into the buffer solution to produce agarose gel.
2. Heat and melt the gel and pour an appropriate amount into a 50mL beaker. Add in Ethidium Bromide until it reaches a concentration of 0.5μg/mL, stir the solution and quickly pour it into a gel-making container and insert 梳子.
3. Keep the solution in room temperature for 30 minutes in order to solidify the gel.
4. Carefully pull out the 梳子 and put the gel into a gel-making container with 1×TAE buffer solution inside. Immerse it completely.
5. Mix the sample with an appropriate amount of 6×Loading Buffer, insert the sampling holes and add to one hole the marker.
6. Turn on the Electrophoresis equipment and set the Voltage to a stable 120 Volts.
7. Stop Electrophoresis when the blue stripe gets close to the middle of the gel.
8. Take pictures of the gel under an ultraviolet lamp.

#### 4. DNA Purification

Operating according to instruction of AxyPrep DNA Gel Recovery Kit

1. Cut the agarose gel that has the gene of interest under UV lamp and calculate the weight of gel (recording the weight of 1.5ml centrifuge tube in advance). The weight is used as a standard gel volume (e.g. 100mg= 100μl ).
2. Add 3 standard gel volume of Buffer DE-A, heat it at 75℃ after mixing, and blend every 2 to 3 minutes until the gel is completely melted.
3. Add Buffer DE-B equivalent to 0.5 volume of Buffer DE-A and mix them uniformly.
4. Transfer the mixed solution in step 3 into preparation tube (placed in a 2ml centrifuge tube provided in the kit). Centrifuge for 1 minute in 12000×g and throw away the filtrate.
5. Place the preparation tube back in the 2ml centrifuge tube. Add 500μl of Buffer W1, centrifuge for 30 seconds in 12000×g and throw away the filtrate.
6. Place the preparation tube back in the 2ml centrifuge tube. Add 700μl of Buffer W2, centrifuge for 30 seconds in 12000×g and throw away the filtrate. Clean with 700μl of Buffer W2 in the same way and centrifuge for 1 minute in 12,000×g.
7. Place the preparation tube back in the 2ml centrifuge tube and centrifuge for 1 minute in 12,000×g.
8. Place the preparation tube back in the 1.5ml centrifuge tube provided in the kit. Add 25 to 30μl Eluent or deionized water in the middle of the preparation membrane, standing in room temperature for 1 minute. centrifuge for 1 minute in 12,000×g and elute the DNA.

#### 5. Plasmid Extraction

The following steps are based on the instruction of plasmid mini prep kit from Generay, Shanghai.

1. Equilibrium of DNA adsorption column: Add 200μl buffer CBS into Plasmid Recovery Column, centrifuge it at 12,000 rpm for 1 min, and discard the waste liquid.
2. According to the growth of bacterial liquid, prepare the liquid cultivated in 2～4ml 2YT or LB, centrifuge it at 12,000 rpm for 1 min, and discard the upper clear liquid.
3. Add 250μl Solution I, and fully suspend the bacteria. Attention: Do not leave small bacteria masses, or the dissociation will be affected, which will lead to a smaller amount and a lower quality of plasmids. Check whether Solution I is added with RNase A.
4. Add 250μl Solution II, and gently invert 6-8 times to dissociate until the liquid becomes clear and albumen-like. This step should not take more than five minutes. If the liquid appears not to be clear, the bacteria may be too much to dissociate, so the amount should be decreased.
5. Add 350μl Solution III, and gently invert 8-10 times. Put them in room temperature for 2-5 min, and then centrifuge them at 12,000 rpm for 10 min.
6. Transfer the upper clear liquid in step 5 into Plasmid Recovery Column. Be careful that you should not transfer the sediments. Centrifuge them at 12,000 rpm for 1 min, take the Plasmid Recovery Column out, and discard the waste liquid of the collection tubes.
7. Replace the adsorption column into the collection tube, add 500μl W1 Solution, centrifuge them at 12,000 rpm for 1 min in room temperature, and discard the waste liquid.
8. Replace the adsorption column into the collection tube, add 600μl Wash Solution, centrifuge them at 12,000 rpm for 1 min in room temperature, and discard the waste liquid.
9. Repeat step 8.
10. Discard the waste liquid, centrifuge them at 12,000 rpm for 2 min, eliminate Wash Solution, and put them in room temperature with the lid opened for several minutes to dry out rinse buffer left.
11. Add Plasmid Recovery Column into clean 1.5ml Eppendorf tubes, add 50～100μl Elution Buffer in the middle of its membrane, put them in 37℃ for 2 min, and centrifuge them at 12,000 rpm for 2 min. The liquid in the Eppendorf tubes is the wanted plasmid.
12. Prepare 1～3μl of it to implement gel electrophoresis. The purified DNA can used in the experiments later or stored in the -20℃ refrigerator.

#### 6. Enzymatic system (20μL)

|  |  |
| --- | --- |
| Plasmid | X μL |
| Universal buffer（10×） | 2.0 μL |
| restriction enzyme A | 1.0 μL |
| restriction enzyme B | 1.0 μL |
| ddH2O | up to 20μL |

The amount of plasmid added in is adjusted according to its concentration. If a large number of plasmids are needed for digestion, the system can be expanded appropriately. Double-enzyme digestion requires the selection of a buffer shared by both restrictive enzymes.

#### 7. Recycling of agarose gel containing plasmid digestion fragments

1. Use scalpel to cut agarose gel containing plasmid digestion fragments under ultraviolet light.
2. Put the cut agarose gel into the 1.5ml centrifuge tube. If the weight of the gel is more than 0.3g, then repeat the procedure.
3. Add 400 μl binding solution, mix evenly and heat the tube at 55℃, continue mixing until the gel melts completely.
4. Add in 30 ml magnetic beads, after shaking and mixing put it in room temperature for 2 minutes, then centrifuge at 6000 rpm for 5 seconds.
5. Add in 600μl washing solution, shake it with fast tempo for 10 seconds, then centrifuge at 6000 rpm for 5 seconds.
6. Add in 75% ethanol solution, shake it with fast tempo for 10 seconds, then centrifuge at 6000 rpm for 5 seconds.
7. Repeat procedure 6.
8. Centrifuge at 10000 rpm for 1 minute. Completely get rid of the invalid portion. Open the centrifuge tube and put it at 55℃ for 5 minutes
9. Add in 25～100μl ddH2O, shake it with fast tempo for 10 seconds. Put it in room temperature for 2 minutes and centrifuge at 10000 rpm for 1 minute. Recycle the invalid portion.

#### 8. Connecting Plasmids

**Operating according to the manual in UniFusion MultiS One Step Cloning Mix**

Linearization of the carrier

We get the linearized carrier by cutting with enzyme or reverse PCR.

Getting the Insert

When connecting single sequences, we take the 15-20bp sequence at the end of the linearized carrier as the homologous sequence, and add it respectively to the 5’ end of the specific genes of the amplifying primer in both forward and backward direction. from which we get the insert with the homologous sequence; When connecting multiple sequences, the homologous sequence must be added to the 5’ side of the amplifying primer to ensure that there is a 20bp homologous sequence between each product and the linearized carrier

Reconstructing Reaction:

The most suitable amount of cloning carrier to use in this system is 0.03pmol, while that of insert is 0.06pmol. (we use 1-3 inserts, the ratio of which to the carrier is 2:1). The corresponding mass of DNA can be estimated with the following formulas:

**N(Cloning Carrier) = [0.02****×****The number of nucleobases in the cloning carrier****] ng (0.03pmol)**

**N(Insert) = [0.04****×****Nucleobases in the insert****] ng(0.06pmol)**

Calculate the amount of DNA needed for the reaction. To ensure the precision of sample adding, the linearized carrier and insert can be diluted before preparing the system and the amount of added sample in each group should be no less than 1μl.

Prepare the following reaction system on ice:

|  |  |
| --- | --- |
| Component | Reaction |
| Linearized Carrier | 20 ng/kb |
| Insert | 40 ng/kb |
| 2×UniFusion MultiS Mix | 10 μl |
| ddH2O | to 20 μl |

X/Y Find the amount needed using the equation.

After completing the preparation, use a pipette to gently mix the solution and avoid creating bubbles. Let it react for 30 minutes under 37℃, after which the reaction tube should instantly cooled in ice bath for 5 minutes. The reagent can then directly transform, or be stored under -20℃ and unfreezed when needed.

#### 9. Cultivation and Restoration of E. coli

E. coli is incubated to LB liquid medium and shook at 37°C overnight. For strains with plasmid, antibiotics (such as 100µg/ml ampicillin, 100g/ml kanamycin and 50µg/ml chloromycetin) should be added into the medium during cultivation to ensure that the plasmid does not lose during the process.

Regarding the restoration during short period, fresh LB solid medium plate is restored in 4° C. Concerning the restoration during long period, cryopreservation using glycerol is needed, which means that the fresh E. coli bacteria solution is added into 40% glycerol with same volume, the mixture is equally blended and stored at -20° C or -80°C.

See 1、Growing overnight cultures and 2、Glycerol stock preparation

#### 10. Preparation of E. coli competent cell

1. Select a single bacteria colony freshly grown on the plate and put it into a tube containing 2mL of LB liquid medium. Cultivate it overnight at 37 °C under the spinning speed of 250 r/min.
2. Transfer 1mL of mixture into 250mL Erlenmeyer flask containing 100 mL of LB liquid medium. Cultivate it at 37 °C under the spinning speed of 250 r/min until the OD600 value of the mixture is between 0.5 and 0.6.
3. Put the Erlenmeyer flask containing bacteria solution quickly onto the ice for 30 min.

Before the next step of the experiment, cool the sterilized 0.1mol/L CaCl2 solution, 0.1mol/L CaCl2-15% glycerol solution, centrifuge tube and the centrifuge to 4 °C.

4. Split the bacteria solution and charge it into 50mL sterilized centrifuge tubes. Centrifuge them at 4 °C, 6000rpm for 5 min.
5. Abandon the clear liquid at the top. Wash the sedimentary bacteria using precooled 0.1 mol/L CaCl2 twice.
6. Centrifuge the tube at 4 °C, 6000rpm for 5 min. Abandon the clear liquid at the top. Add 1mL of 0.1mol/L CaCl2-15% glycerol solution and put the tube into the vortex. Transfer 100µL of the mixture into each 1.5mL Eppendorf tube quickly and store them at -80 °C.

See 13、Preparation of competent cells

#### 11. Transformation of E. coli

1. Blend 2μl of plasmid or 10μl of recombinant DNA with 100ul of competent cell. Softly flick the tube walls to mix them, then ice bath over 30 minutes.
2. Thermal excitation at 42℃for 90 seconds. Then, quickly place the centrifugal tubes on ice for 5 minutes.
3. Pipete 800μl of LB liquid medium into each tube and mix them. Shake the bacteria in shaker at 37℃, 130r/min for 45 minutes.
4. For transformation of Plasmid, take 100μl liquid bacteria and evenly spread plate (containg antibiotic). For trasformation of recombinant DNA,centrifugate liquid bacteria 5000r/min for 3 minutes to collect the thallus. Resuspend E.coli with 200μl liquid medium and then spread the bacteria on plates.
5. Place plates upside down and culture E.coli for overnight.

#### 12. Colony Identification

Bacteria colony PCR

1. Having oberserved the grow of single bacteria colony, split 600μl of LB liquid medium (containing antibiotic) into each 1.5mlEppendorf tube in clean bench. Use sterial small pipete tips to pick single colonies of bacteria into Eppendorf tube and mix it with LB liquid medium. Shake the bacteria in shaker at 37℃, 250r/min over 6 hours.
2. Pipete 2μl of liquid bacteria into ColonyPCR reaction system as PCR template. Conduct bacteria liquid PCR.
3. Carry out agarose gel electrophoresis of PCR products and observe whether there are target bands.Transfer liquid bacteria of positive results to LB liquid medium (containing antibiotic) at 37℃, 250r/min overnight. Pipete 300μl of the liquid bacteria for DNA sequencing.

#### 13. The extraction and testing of carotenoids

1. Separate the mycelium containing CrtN gene and cultivate them overnight at 37℃,250 r/min.
2. Transfer 10% of the overnight cultivated liquid into the test tube containing 3ml TB medium, and cultivate it at 37℃ and 250 r/min until OD600 reaches 0.6-0.9, then induce with IPTG of the final concentration of 0.1mM.
3. Cultivate for 48 hours at 37℃,250 r/min.

Collect the mycelium by centrifuge at 8000rpm for 1 minute, clean it with 2ml PBS, then centrifuge and get rid of the invalid portion.

4. Add 1ml acetone into the collected mycelium, rotate it for 3 minutes, then centrifuge at 12000rpm for 10 minutes.
5. Test the invalid portion under OD470nm.

#### 14. E. coli Shake-Flask fermentation

1. Pick a single colony from the plate to 3ml of culture with LB, grow overnight in 37℃ at 250r/min.
2. Transfer them at 10% inoculation amount into 250ml triangular flakes with 20ml TB in 37℃, and cultivate them at 250r/min until the OD600 reaches 0.6-0.9. Add 0.1mM IPTG in 30℃, cultivate it at 180r/min for 96h.
3. After strains without IPTG are transferred into TB in 37℃ for 12h at 180r/min, adjust the temperature to 30℃, and cultivate them at 180r/min for 96h.

#### 15. Squalene Extraction and Characterization

Sample processing:

1. Decant the fermentation broth into a 50ml centrifuge tube, centrifuge for 1 minute in 8000rpm and remove the supernate.
2. Clean with 1% NaCl(w/v) solution and remove the supernate.
3. Add 1ml of 1% NaCl solution to resuspend the thallus. Add 5ml of acetone. After eddying uniformly, break the thallus with ultrasound for 10 minutes.
4. Add 10ml of 1% NaCl solution and 2ml of hexane, centrifuge for 30 minutes in 3300rpm, and transfer the hexane phase into a 2ml EP tube and place it in a vacuum drying oven to remove the hexane.

#### 16. High Performance Liquid Chromatography Analysis

1. Sample preparation: transfer 400L of methanol and THF in a ratio of 4 to 6 into a sample tube that has been dried in vacuum, eddying for 1 minute. It can be used for HPLC analysis after filtrating through a 0.22m filtration membrane.
2. HPLC condition:

moving phase: acetonitrile: THF: methanol is 58: 7: 35(v/v)

flow velocity: 1mL/min

column temperature: 30℃

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* [Synthase Screening](#screening)
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Squalene production by E.coli

Squalene production by E.coli

#### Screening of highly active squalene synthase

Because the precursors of squalene synthesis, farnesyl diphosphates (FPP), are available in E. coli, and thus, the production of squalene in E. coli is achieved simply by expressing squalene synthase (SQS). In this study, in order to obtain squalene synthase with higher activity, we cloned the squalene synthase from Y. lipolytica (YSS), S. cerevisiae (NSS), B. subtilis (KSS), and the truncated squalene synthase from human being (thSQS). The membrane-binding domains of hSQS (30 residues of the N-terminus and 47 residues of the C-terminus) were truncated based on the previous report, while YSS, NSS and KSS were expressed as the full-length sequence.

It was reported that the dehydrosqualene desaturase CrtN from Staphylococcus aureus converts squalene into yellow carotenoid pigments (63), permitting the colorimetric detection of squalene produced in the cell. In order to screen out squalene synthase with high activity rapidly, the SQS genes and CrtN gene were sub-cloned into the MCS1 and MCS2 of plasmid pETDuet-1respectively, yielding pETDuet-1T7-YSS-2T7-CrtN(pET-YN), pETDuet-1T7-NSS-2T7-CrtN(pET-NN), pETDuet-1T7-KSS-2T7-CrtN(pET-KN), pETDuet-1T7-thSQS-2T7-CrtN(pET-thN).

Plasmids with different SQS genes and CrtN gene were transferred with plasmid p35151 (Figure. 1C) into E. coli BL21 (DE3), and strains N1 (P35151/ pET-KN), N2 (P35151/ pET-NN), N3(P35151/ pET-thN) and N4(P35151/ pET-YN) were constructed. After 48 hours of fermentation, carotenoid pigments detection (Figure. 1B) was conducted on these strains. The results showed the pigmentation level of strain N4 expressing YSS was higher than that of strains expressing NSS, KSS and thSQS, indicating that YSS from Lysergic yeast has higher activity. So we decided to use YSS to construct the squalene synthesis pathway in E. coli.

![](https://static.igem.org/mediawiki/2019/5/5e/T--SEFLS_Shanghai--results-fig1.png)

Figure.1 A. sequence structure of four plasmids B. carotenoid pigments detection C. sequence structure of plasmid 35151

#### The Effect of Temperature on Squalene Accumulation

Because five components (tHMGR, HMGS, MK, PMK, PMD) of the mevalonate pathway (P35151) used here originated from yeast, we sought to determine the optimal temperature for these enzymes. Strain N4 was cultured at 30℃ or 37℃ to compare the pigmentation level. The results show that the pigmentation level was very low at 37℃, indicating that the optimal temperature for these enzymes was 30℃. Therefore, we chose 30°C as the fermentation temperature of the strains.

#### Functional Expression of YSS

In order to synthesize squalene in E. coli, the YSS gene was sub-cloned into plasmid pETDuet-1, yielding pET-YSS. Co-transformation of the resultant plasmid pET-YSS with p35151 (strain H1) resulted in squalene production of 18.9 mg/L (Figure. 1B). In order to further confirm that the fermentation product is squalene, we also used GC-MS to analyze its structure.

![](https://static.igem.org/mediawiki/2019/9/9c/T--SEFLS_Shanghai--results-fig2.png)

Figure2. A. relationship between peak area and squalene concentration B. result of co-transformation C. GC-MS analysis

#### Efficient Squalene Production

We had proposed three different strategies for engineering an efficient E. coli producer of squalene

# Increase in the supply of precursor FPP

In order to increase the supply of precursor FPP, we overexpressed the gene idi and ispA. Genes idi and ispA were added to the plasmid pET-YSS, yielding pET-IAY. Co-transformation of the plasmid pET-IAY with p35151 resulted in squalene production of 69.3 mg/L (strain H2), an approximately 3.7-fold increase compared to BL21(DE3) harboring pET-YSS and p35151(Figure. 3). This result indicated that the overexpression of idi and ispA increased the supply of precursor, and thus increasing the yield of squalene.

# Increase in the supply of precursor IPP and DMAPP

The MVA and MEP pathways have been the targets of many metabolic engineering efforts to increase the supply of IPP and DMAPP in host microorganisms for improved terpenoid production.

# Optimization of MEP Pathway

In order to optimize MEP pathway, we overexpressed the key enzymes Dxs, IspG and IspH (Go to Design) in the MEP pathway. It was suggested that balanced activation of ispG and ispH could push the carbon flux away from not only HMBPP but also DXP, thus leading to a more efficient MEP pathway. We did balanced activation by constructing genes ispG and ispH into plasmids with different copies and promoters of different strength.

These genes were respectively constructed into plasmids with different copies. The genes dxs and ispG were placed on the backbone of low-copy plasmid pBBR1MCS-2, and the promoter was medium strength lac promoters, to construct plasmid pBBR1MCS-2-dxs-ispG (pMEP-DG). Genes ispH, idi, and ispA were placed on the backbone of high-copy plasmid pETDuet-1, and the promoter was strong promoter T7 to construct plasmid pETDuet-1-1T7-IspH-Idi-IspA-Yss (pET-HIAY).

P35151 was combined with plasmids pMEP-DG and pET-HIAY containing the key MEP enzyme genes. They were then co-transferred into E. coli BL21 (DE3) to construct strain H5. We also combined p35151 with pET-HIAY, p35151, pMEP-DG and pET-IAY to construct strains H3 and H4.

Name of Strains

Combination

Squalene Yield (mg/L)

H1

p35151/pET-YSS

18.9

H2

p35151/pET-IAY

69.3

H3

p35151/pET-HIAY

50.4

H4

p35151/pMEP-DG/ pET-IAY

298.2

H5

p35151/pMEP-DG/ pET-HIAY

472.3

![](https://static.igem.org/mediawiki/2019/d/d2/T--SEFLS_Shanghai--results-fig3.png)

Figure.3 construction of strains H1, H2 & H3

Comparing the squalene yield, we found that on the basis of overexpressing genes idi and ispA, only overexpressing gene ispH will decrease the squalene yield. It was speculated that the decline was because the overexpressed gene ispH consumed large amounts of intermediates HMBPP, which led to an insufficient supply of HMBPP for IspH to react, thus hindering the metabolic flow in MEP pathway.

When Dxs and IspG were overexpressed, the squalene yield increased by 4.3 times. We speculated that the overexpressed gene ispG was constructed on low-copy plasmids and was controlled by promoters of medium strength, so it didn’t produce large amounts of harmful intermediate HMBPP. And overexpressing Dxs promoted the metabolic flow, increasing the squalene yield.

Meanwhile, after overexpressing Dxs, IspG and IspH, the squalene yield was 1.6 times higher than only overexpressing Dxs and IspG, which meant that different overexpression combination of IspG and IspH could lead to a more efficient MEP pathway, thus increasing the squalene yield.

# Optimization of MVA Pathway

Plasmid P35151 includes 6 genes (AtoB, HMGS, tHMGR, MK, PMK and PMD) in the MVA pathway and gene idi. These seven genes were integrated into a single operon. We optimized according to the MVA pathway in the strains producing fannene in the report, introducing MVA pathway gene included in plasmid P35151 separated in two parts on two plasmids to construct pBBR1MCS-1(p15A)-AtoB-HMGS-tHMGR (pMVA1) and pBBR1MCS-2-MK-PMK-PMD-Idi (pMVA2).

pMVA1 containing the first three genes atoB, HMGS and tHMGR in the MVA pathway took the plasmid pBBR1MCS-1 as the skeleton carrier, the promoter was lac of medium strength, and the replicon was replaced by p15A. pMVA2 containing the last three genes in the MVA pathway and gene idi took the plasmid pBBR1MCS-2 as the skeleton carrier, the promoter was lac of medium strength, and the replicon was pBBR1 oriV. The seven genes on plasmid pMVA1 and pMVA2 all came from gene P35151.

Plasmids pMVA1 and pMVA2 were used to co-transform BL21 with pET-YSS or pET-IAY. Strains H6 (pMVA1/pMVA2/pET-YSS) and H7 (pMVA1/pMVA2/pET-IAY) were constructed. Strains H6 and H7 were shaken and fermented at 30℃ for 96h, and the average yield of squalene was 617.8 mg/L for strain H6，974.3 mg/L for strain H7, an approximately 52-fold increase compared to strain H1. The squalene yield of the combination of pMVA1, pMVA2 and pET-YSS was 32.7 times higher than that of the combination of P35151 and DMAPP, indicating that the combination pMVA1 and PMVA2 after reconstruction has effectively improve the supply of precursors IPP and DMAPP. The squalene yield of the combination of pMVA1, pMVA2 and pET-IAY was 1.6 times higher than that of the combination of pMVA1, pMVA2 and pET-YSS, indicating that the overexpression of Idi and IspA has improved the supply of precursors FPP, increasing the squalene yield.

Name of Strains

Combination

Squalene Yield (mg/L)

H6

pMVA1/pMVA2/ pET-YSS

617.8

H7

pMVA1/pMVA2/ pET-IAY

974.3

![](https://static.igem.org/mediawiki/2019/1/19/T--SEFLS_Shanghai--results-fig4.png)

Figure.4 A&B. construction of strains H6 & H7 C. squalene yield comparison

# Replacement of Chassis Cells

In the report whose squalene yield was 230 mg/L, the chassis cell used to produce squalene was E. coli XL1-Blue. Different host strain has influence on the synthesis of target products. As a result, we tried to replace chassis cell BL21 (DE3) with XL1-Blue to observe the effect of different chassis cells on squalene yield.

The current expression carrier promoted by T7 promoter need to be expressed only in strain BL21(DE3). If the chassis is replaced with XL1-Blue, we need to construct genes ispH, idi, ispA and YSS on the high-copy carrier pUC19m whose promoter is lac to construct plasmid pUC19m-Idi-IspA-YSS (pUC-IAY) and plasmid pUC19m-IspH-Idi-IspA-YSS (pUC-HIAY).

p35151, pMEP-DG and pUC-IAY were combined together to co-transform E. coli XL1-Blue to construct strain XH3. The squalene yield was 293.7mg/L. p35151, mMEP-DG and pUC-HIAY were combined together to construct strain XH4. The squalene yield was 504.0mg/L, which was 1.7 times higher than the yield of strain XH3. This indicated that the composite parts containing Dxs, IspG and IspH we constructed can be used in different E. coli chassis cells to optimize MEP pathway and the supply of precursor IPP and DMAPP in the chassis cells.

Name of Strains

Combination

Squalene Yield (mg/L)

XH3

p35151/ pMEP-DG/ pUC-IAY

293.7

XH4

p35151/pMEP-DG/ pUC-HIAY

504.0

XH5

pMVA1/pMVA2/ pUC-IAY

1274

pMVA1, pMVA2 and pUC-IAY were combined together to co-transform E. coli XL1-Blue. The squalene yield was 1.3g/L(Figure. 5), which was 1.3 times higher than strain H7 of chassis cell BL21 (DE3), showing that XL1-Blue was more suitable as the chassis cell to synthesize squalene. Also, this level of squalene production is 5.5-fold of the highest reported value (230 mg/L) for E. coli.

![](https://static.igem.org/mediawiki/2019/5/5a/T--SEFLS_Shanghai--results-fig5.png)

Figure 5. construction of strains XH3, XH4 & XH5 and squalene yield comparison