iGEM 2022 — Team pinghe

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{% block title %}Notebook{% endblock %}
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# Notebook

## 7/8 (online)

1. Laboratory safety training
2. Background research
3. Project discussion

## 7/10

1. Use Salmonella Enterica Genome as PCR template, amplify fadB enzyme through PCR; use pUC57-ter as PCR
   template, amplify gene ter through PCR;
   use pUC57-bktB as PCR template, amplify gene bktB through PCR;
   use *Escherichia coli* genome as PCR template, amplify gene ydi1 through PCR
2. Gel extraction.
3. Double-enzyme digest pETDuet-1 with Nco1/BamH1, gel extraction.
4. Ligate fadB fragment and digested pETDuet-1 vector using one-step cloning method, transfer the recombinant
   plasmid into DH5α competent cells and coat on solid LB medium.
5. Double-enzyme digest pCDFDuet-1 using Nco1/BamH1, extract the gel.
6. Ligate bktB fragment and digested pCDFDuet-1 vector using one-step cloning method, transfer the
   recombinant plasmid into DH5α competent cells and coat on solid LB medium.

## 7/11

1. Inoculate monoclonal colonies of pETD-fadB and pCDFD-bktB into LB medium.
2. Use pETD-EGFP plasmid as PCR template, amplify GFP through PCR, and gel extraction. (F, R primers carry
   homologous recombination sequences)
3. Double-enzyme digest pET28a plasmid using Nco1/BamH1, extract the gel.
4. Mix the GFP fragments and the digested plasmid fragments at a molar concentration ratio of 3:1, and use
   Hieff homologous recombinase to ligate, transfer the recombinant plasmid into DH5α competent cells, and coat
   on a solid LB medium.

## 7/12

1. Extract pETD-fadB and pCDFD-bktB plasmid, and verify whether they are successfully constructed through
   Nco1/BamH1 double-enzyme digest.
2. Inoculate the monoclonal colonies of pET28a-EGFP into LB medium.

## 7/13

1. Extract the pET28a-EGFP plasmid, and verify whether it is inserted into the original plasmid template
   using Nco1/BamH1 double-enzyme digest.
2. Double-enzyme digest pETD-fadB using NdeI/XhoI, gel extraction.
3. Ligate gene ter fragment with the digested pETD-fadB vector using the one-step cloning method, transfer
   the recombinant plasmid into DH5α competent cells, and coat on a solid LB medium.
4. Double-enzyme digest pCDFD-bktB using NdeI/XhoI, gel extraction.
5. Ligate gene ydi1 fragment with digested pCDFD-bktB fragment using the one-step cloning method, transfer
   the recombinant plasmid into DH5α competent cells, and coat on a solid LB medium.

## 7/14

1. Inoculate monoclonal colonies of pETD-fadB-ter and pCDFD-bktB-ydi1 into LB medium.

## 7/15

1. Extract pETD-fadB-ter and pCDFD-bktB-ydi1 plasmid, and verify whether they are inserted into the original
   plasmid template through NdeI/XhoI double-enzyme digest.
2. Use pET28a-EGFP plasmid as PCR template, and amplify 4 different kinds of T7 promotor mutant (C1~C4)
   through PCR.
3. Dpn1 digestion, transfer the recombinant plasmid into DH5α competent cells, and coat on a solid LB
   medium.

## 7/16

1. Inoculate the monoclonal colonies of T7 point mutation plasmids into LB medium.

## 7/17

1. Extract T7 point mutation plasmids, and verify whether the point mutation was successfully constructed
   using sanger sequencing.

## 7/18

1. Transfer the correct pET28a-EGFP\_C1-C4 plasmids to BL21(DE3) competent cells, and coat on a solid LB
   medium.

## 7/19

1. Incubate BL21(DE3) strain containing pET28a-EGFP\_C1-C4 plasmids in 5mL LB medium overnight.

## 7/20

1. 1% transform the cultured medium into 25mL fresh LB medium until OD600=0.5.
2. Induce protein expression using IPTG, and incubate for 3h.
3. Measure OD600 and the fluorescence intensity of GFP to select the optimal mutation.
4. Use pETD-fadB-ter and pCDFD-bktB-ydi1 as PCR templates, amplify the optimal T7 point mutation plasmid
   using PCR.
5. Dpn1 digestion, transfer the recombinant plasmid into DH5α competent cells, and coat on a solid LB medium
   plate.

## 7/21-7/23

1. Inoculate the monoclonal colonies into LB medium.
2. Extract plasmids, and verify whether the point mutation was successfully constructed using sanger
   sequencing.
3. Transform the correct plasmids into BL21 (DE3), coat on a solid LB medium plate.

## 7/24-7/26

1. Inoculate the BL21 (DE3) monoclonal colonies into LB medium, and incubate at 37℃ overnight.
2. 1% transform the cultured medium into 25mL fresh LB medium until OD600=0.5.
3. Induce protein expression using 0.5 mM IPTG, and incubate for 40h.
4. SDS-PAGE gel electrophoresis verification.

## 7/27

1. Send the Bacterial Lysates to the company for HPLC detection of MCFAs.

## 8/1-8/15

1. Collect and analyze HPLC detection data
2. Write wiki and WeChat articles

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{% block title %}Engineering{% endblock %}
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# Engineering

## Introduction

Medium-chain fatty acids (MCFAs) have a favorable safety profile and can be used to treat a variety of
conditions, such as benefits for skin care, weight loss, and cardiovascular use. However, most of the
medium-chain fatty acids in use today are extracted from tropical plants, which is time-consuming and limits
the yield of medium-chain triglycerides. As a result, its yield is much lower than that of long-chain fatty
acids.

In order to overcome this limitation, we used synthetic biology strategies to conduct experiments on the four
core enzymes of the reverse β-oxidation cycle, namely fadB, ydiI, ter, and bktB. This was then applied to the
MCFAs synthesis pathway in *E. coli* to increase the production of MCFAs.

## How we design our plasmids

The production of medium-chain fatty acids (MCFAs) can be increased by overexpressing key enzymes in the
reversal of the β-oxidation cycle. We use the four key enzymes, bktB, fadB, ter, and ydiI to construct the
reverse β-oxidation cycle and form the MCFAs synthesis pathway in *E. coli*. In this way, we can improve
the
yield of the production of MCFAs.

Because the T7 promoter has a strong ability in translation and usually be used as protein expression, we
choose the pETDuet-1 vector and pCDFDuet-1 vector, with T7 promoter respectively, to express our target
enzymes bktB, fadB, ter, and ydiI to develop r-BOX cycle. To achieve this, we inserted genes fadB and ter into
pCDFDuet-1vector, and inserted genes bktB and ydiI into pCDFDuet-1vector, transferred the recombinant plasmid
into *E. coli* BL21(DE3) for protein expression (Figure 1).

![](https://static.igem.wiki/teams/4284/wiki/engineering/t-pingehe-engineering-01.png)

Figure 1. the workflow of this project (Junjun Wu , et al., 2019)

## How we build our plasmid

In order to construct our plasmids, we amplify all four enzymes with corresponding templates by PCR.
Salmonella enterica genome was used as the template to amplify gene fadB, the plasmid pUC57-ter was used as
the template to amplify gene ter, the plasmid pUC57-bktb was used as the template to amplify gene bktB, and
the genome of *E. coli* MG1655 was used as the template to amplify gene ydiI (Figure 2).

![](https://static.igem.wiki/teams/4284/wiki/engineering/t-pingehe-engineering-02.jpg)

Figure 2. Gel electrophoresis results of target gene fragments.

Line 1: DNA *marker*
Line 2: *bktB* 1176bp
Line 3: *ydiI* 501bp
Line 4: *fadB* 2280bp
Line 5: *ter* 1299bp

In Figure 2 we can find that there were four clear DNA bands, indicating that genes bktB, ydiI, fadB and ter
were successfully amplified by PCR.

Then we used a one-step cloning method to ligate the gene fadB into the NcoI and BamHI sites of the pETDuet-1
vector. Then we transformed the recombinant plasmid into *E. coli* DH5α competent cells. We inoculated
the
correct strain and extracted the plasmid pETDuet1-fadB. Then we inserted gene ter into the NdeI and XhoI sites
of pETDuet1-fadB through a one-step cloning method. The recombinant plasmid pETDuet1-fadB-ter was verified by
NcoI/HindIII and NdeI/XhoI, respectively (Figure3 line2, 4).

Meanwhile, we also used a one-step cloning method to ligate the gene bktB into the NcoI and BamHI sites of the
pCDFDuet-1 vector. Then we transformed the recombinant plasmid into *E. coli* DH5α competent cells. We
inoculated the correct strain and extracted the plasmid pCDFDuet1-bktB. Then we inserted gene ydiI into the
NdeI and XhoI sites of pCDFDuet1-bktB through a one-step cloning method. The recombinant plasmid
pCDFDuet1-bktB-ydiI was verified by NcoI/HindIII and NdeI/XhoI, respectively (Figure3 line1, 3).

![](https://static.igem.wiki/teams/4284/wiki/engineering/t-pingehe-engineering-03.png)

Figure 3. Digestion verification of pETDuet1-fadB-ter and pCDFDuet1-bktB-ydiI.

M: DNA marker
1: fadB fragment and carrier fragment
2: ydiI fragment and carrier fragment
3: ter fragment and carrier fragment
4: bktB fragment and carrier fragment

We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing
comparison results showed that there were no mutations in the ORF region (Figure 4.), and the plasmids were
successfully constructed. So far, we have successfully obtained the two recombinant plasmids.

![](https://static.igem.wiki/teams/4284/wiki/engineering/t-pingehe-engineering-04.jpg)

Figure 4. The sequencing blast results of the recombinant plasmids.

A. Sequencing and comparison of pETD-*fadB*
B. Sequencing and comparison of pCDF-*ter*
C. Sequencing and comparison of pCDF-*bktB*
D. Sequencing and comparison of pETD-*ydiI*

## How we test our r-BOX cycle

### a) Protein expression and purification

In order to obtain the four enzymes, we transferred the recombinant plasmids, pETDuet1-fadB-ter and
pCDFDuet1-bktB-ydiI, into *E. coli* BL21(DE3), inoculated the correct colony in the LB culture medium
and
added IPTG to induce protein expression when the OD600 reached 0.5. After overnight induction and culture,
we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we
used nickel column purification to purify the enzymes we wanted, and detected the proteins by SDS-PAGE
(Figure 5).

![](https://static.igem.wiki/teams/4284/wiki/engineering/t-pingehe-engineering-05.png)

Figure 5. SDS-PAGE analysis of bktB, ydiI, fadB, ter.

Line 1: *marker*
P2: *bktB*
P3: *ydiI*
P4: *fadB*
P5: *ter*
P1: fadB, ter, bktB, ydiI
pCDFDeut-1: negatice control

Four clear protein bands can be seen in figure 5, while there is no corresponding band in the control,
indicating the successful expression of bktB, ydiI, fadB and ter.

### b) MCFAs fermentation test

To confirm the ability of the platform we developed in BL21(DE3) with the genes r-BOX cycle (bktB, ydiI,
fadB and ter), can produce MCFAs, we detected the yield of MCFAs through HPLC.

## Results of functional test of strain

We co-transformed the recombinant plasmids, pETDuet1-fadB-ter and pCDFDuet1-bktB-ydiI, into BL21(DE3), and
inoculated the successfully transformed strain into LB medium overnight at 37℃. Then transferred the cultured
medium into 25 mL fresh LB culture medium and make the initial OD600 equal to 0.1 and incubated at 37℃ 200rpm.
Added Glucose to the medium when OD600 was about 0.5, IPTG was also added to induce the expression of R-box at
a final concentration of 1mM. After culturing for 40h, we collected 6 mL of the culture medium, collected the
cells, and ultrasonic crushing was performed, centrifuged, and collect the supernatant. Mixed the supernatant
with 2-Bromoacetophenone and Triethylamine, incubated at 50℃ for 4h, and then use HPLC to detect the yield of
MCFAs (Figure 6).

![](https://static.igem.wiki/teams/4284/wiki/engineering/t-pingehe-engineering-06.jpg)

Figure 6. HPLC profiles of BL21(DE3) strain expressing r-BOX.

A: MCFAs standard
B: BL21\_ *bktB- ydiI- fadB-ter*

As shown in Figure 6, After the HPLC analysis of C4-C12 fatty acid standards and BL21\_bktB-ydiI-fadB-ter, at
the same location of the peak as the standard MCFAs, B represented the successful production of 1.08 g/L MCFAs
by strain BL21\_ *bktB- ydiI- fadB-ter*. So that our platform for producing MCFAs was successfully
established.

## How we learn from our project

The results showed that the overexpression of the β-oxidation reversal composed of four enzymes, fadB, ydiI,
ter, and bktB, can effectively increase the production of MCFAs in *E. coli*. In this case, a higher
yield of
products can be made through our genetic engineering. We believed that our engineered strain could be used for
future research or even be applied for industrial production.

Because of the important effect on human daily life, if we can produce more MCFAs in the future, we believe
that it will provide people to live in a healthier way.

## Reference

1. Wu J, Wang Z, Duan X, Zhou P, Liu P, Pang Z, Wang Y, Wang X, Li W, Dong M. Construction of artificial
   micro-aerobic metabolism for energy- and carbon-efficient synthesis of medium chain fatty acids in
   Escherichia coli. Metab Eng. 2019 May;53:1-13. doi: 10.1016/j.ymben.2019.01.006. Epub 2019 Jan 24. PMID:
   30684584.

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# Experiments

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# Results

## Experimental results of synthesis of medium-chain fatty acids by reverse fatty acid β-oxidation cycle

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