iGEM 2022 — Team ecnuas

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# Notebook

## 7/1

Team ice-breaking.

Team discussion（Team name, uniform, logo...）.

![](https://static.igem.wiki/teams/4281/wiki/notebook/t-ecnuas-notebook-01.jpg)

## 7/2

Laboratory safety training.

Configuring the culture medium.

Inoculation of the *Streptomyces rapamycinicus*, 30℃.

Inoculation of the strain containing plasmid pKC1139.

![](https://static.igem.wiki/teams/4281/wiki/notebook/t-ecnuas-notebook-02.jpg)

## 7/3

Design the questionnaires.

Extract plasmid pKC1139 using a plasmid extract kit.

![](https://static.igem.wiki/teams/4281/wiki/notebook/t-ecnuas-notebook-03.jpg)

## 7/4

Team collaboration.

![](https://static.igem.wiki/teams/4281/wiki/notebook/t-ecnuas-notebook-04.jpg)

Amplify target DNA fragments by PCR.

PCR result:

Line 1, 3, and 5 are upstream homologous gene fragments, and line 2, 4, and 6 are downstream homologous gene
fragments.

![](https://static.igem.wiki/teams/4281/wiki/notebook/t-ecnuas-notebook-05.jpg)

Line 1, 4 and 7 are plasmid pKC1139.

Line2,3,5,6,8,9,10 are double enzyme digested plasmid pKC1139 (EcoRI/HindIII).

Construct recombinant plasmids by Homologous reorganization.

## 7/5

Verification of the recombinant plasmids pKC-M271\_ 14685/ M271\_ 14690through colony PCR.

Inoculation of the strain containing correct plasmids.

## 7/6

Extract the plasmids pKC-M271\_ 14685/ M271\_ 14690 with a plasmids extraction kit.

Double enzyme digestion verification.

![](https://static.igem.wiki/teams/4281/wiki/notebook/t-ecnuas-notebook-06.jpg)

Line 1,3,5 are recombinant plasmid pKC-M271\_ 14685/ M271\_ 14690.

Line 2,4,6 are recombinant plasmid pKC-M271\_14685/ M271\_14690 digested with EcoR I/Hind III

## 7/7

Expert interview.

Transfer plasmids pKC-M271\_ 14685/ M271\_ 14690 into ET12567/pUZ8002 competent cells, and coat to LB plate
containing antibodies, 37℃ incubate overnight.

![](https://static.igem.wiki/teams/4281/wiki/notebook/t-ecnuas-notebook-07.jpg)

## 7/8

Take fixed makeup photos.

Pick up single colonies and inoculate them in 4ml fresh culture medium,37℃ 220rpm overnight.

## 7/9

1. Transfer 1ml strain suspension into 50ml fresh culture medium, when OD600 is around 0.4-0.6, centrifuge
and discard the medium, wash it twice with LB (no antibody contained) and resuspended it with 500ul
culture medium.

2. Collect the *Streptomyces rapamycinicus* spore through 2×YT culture medium which was incubated on the
plate.

3. Mix the *E. coli* suspension from step (1) and the spore suspension from step (2), coat onto M-ISP4
solid medium, and incubate at 30°C.

## 7/10

After incubated on the M-ISP4 solid medium for 16h, add antibodies, and incubate at 30℃ for 7 days.

## 7/17

PCR identification of colony.

Screening for single cross-over strains.

Filtrate single cross-over strains.

![](https://static.igem.wiki/teams/4281/wiki/notebook/t-ecnuas-notebook-08.jpg)

## 7/23

Carry out educational activities.

Expert interview.

Screening double cross-over strains.

The correct strain was streaked on the oat solid medium and cultured at 30°C for 7 days

![](https://static.igem.wiki/teams/4281/wiki/notebook/t-ecnuas-notebook-09.jpg)

## 7/29

Expert interview.

Writing implementation.

## 7/30

Fermentation culture of *Streptomyces rapamycinicus*.

## 8/6

Pick up colonies and inoculate them in 3ml fresh fermentation culture medium,28℃ 220rpm overnight.

## 8/7-8/18

Transfer 3ml strain suspension into 30ml fresh fermentation culture medium, culture at 28℃ 200rpm, and
collect samples at 5d, 7d, 9d, and 11d.

Rapamycin production estimation by HPLC.

![](https://static.igem.wiki/teams/4281/wiki/notebook/t-ecnuas-notebook-10.jpg)

## 8/19

Write wiki.

Make a presentation video.

Prepare for presentation.

![](https://static.igem.wiki/teams/4281/wiki/notebook/t-ecnuas-notebook-11.jpg)

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# Engineering

## Introduction

Organ transplantation faces the problem of immune rejection while giving patients a second life. Rapamycin is
a new type of macrolide antibiotic, and it is also a widely used clinical drug for the treatment of immune
rejection, which can greatly improve the survival rate of transplanted organs after surgery. But the drug is
produced at a low level and is expensive.

In our project we optimized the metabolic regulation network of rapamycin biosynthesis by knocking out the
two-component system encoding gene *M271\_14685/M271\_14690* in *Streptomyces* *rapamycinicus*,
thus bringing greater value to clinical treatment and bringing good news to patients with organ
transplantation.

## Design

In this experiment, we amplified the 1000bp upstream and downstream gene fragments of
*M271\_14685/M271\_14690*, which are two-component systems encoding genes in *Streptomyces* *rapamycinicus*,
ligated them into the plasmid pKC1139, and then transformed it into the host strain *Streptomyces* *rapamycinicus*.
Through homologous recombination, single-crossover strains and double-crossover strains were obtained, and the
target gene *M271\_14685/M271\_14690* was knocked out to finally obtain mutant strain *ΔM271\_14685/M271\_14690*
(Fig1).

![](https://static.igem.wiki/teams/4281/wiki/description/t-ecnuas-description-01.jpg)
Figure 1. The two-component systems working diagram

## Build

A gene knockout plasmid (pKC*M271\_14685/M271\_14690*) was constructed (Fig. 2). The plasmid is used to
knock out the two-component system encoding gene *M271\_14685/M271\_14690* in *Streptomyces* *rapamycinicus*.
The construction was confirmed by DNA sequencing.

![](https://static.igem.wiki/teams/4281/wiki/engineering/t-ecnuas-engineering-02.png)
Figure 2. Schematic map of the plasmid

We designed the program by inserting the *M271\_14685/14690* upstream homology arm gene into HindIII and
EcoRI sites of the pKC1139 vector. In order to build our plasmids, we amplified the gene fragments from the
genome of *Streptomyces* *rapamycinicus* NRRL 5491 by PCR (Figure 3), double-enzyme digestion, and
ligase to pKC1139 carrier.

![](https://static.igem.wiki/teams/4281/wiki/engineering/t-ecnuas-engineering-03.png)

Figure 3: Gel electrophoresis diagram.

Line 1,3,5 are *M271\_14685/14690* upstream homology arm gene, 1208bp.
Line 2,4,6 are *M271\_14685/14690* downstream homology arm gene, 1144bp.

In Figure 3, a clear and single DNA band at 1kp can be seen, indicating that the upstream and downstream
homology arms of *M271\_14685/M271\_14690* were successfully amplified by PCR.

![](https://static.igem.wiki/teams/4281/wiki/engineering/t-ecnuas-engineering-04.png)

Figure 4: Gel electrophoresis diagram.

Line 1,3,5: recombinant plasmid pKC-*M271\_14685/M271\_14690*, 10320bp.
Line 2,4,6: recombinant plasmid pKC-*M271\_14685/M271\_14690* digested with EcoRI/HindIII, 7962 bp, 2358 bp.

To verify if the recombinant plasmid is correct, we did double-enzyme-digestion. It can be seen from the
Figure 4 that the size of the plasmid we constructed is correct, and the identification results of double
enzyme digestion are also correct.

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, and the plasmid was successfully
constructed.

To construct the engineering strain, we firstly transferred the recombinant plasmid into ET12567/pUZ8002
competent cells and screened the correct strain through 3 antibodies, and cultured it in the liquid medium.
Then co-cultured the *E.coli* with *Streptomyces* *rapamycinicus* and screened for the
cross-over strain.

It can be seen from Figure 5 that the gene fragments of the engineered bacteria we constructed were
successfully exchanged compared with the negative control. That is, the engineered bacteria that knocked out
the *M271\_14685/M271\_14690* gene were successfully obtained.

![](https://static.igem.wiki/teams/4281/wiki/engineering/t-ecnuas-engineering-05.png)

Figure 5. Gel electrophoresis diagram. Line 1: double cross-over strain, 326bp, correct.

Line 2: control strain: *Streptomyces* *rapamycinicus* NRRL 5491, 3475bp.

## Test

Co-cultured *Streptomyces* *rapamycinicus* NRRL 5491 with *ΔM271\_14685/M271\_14690* in the
fermentation medium, we collected the sample respectively at 5d,7d,9d, and 11d. We mixed 0.5 mL samples with
the same volume of methanol and tested the yield of rapamycin after centrifuge. To identify the correct peek
of rapamycin, we use the standard rapamycin as positive control. It can be seen from Figure 8 that the *△M271\_14685/m271\_14690*
detected more rapamycin at the same time as standard rapamycin.

![](https://static.igem.wiki/teams/4281/wiki/engineering/t-ecnuas-engineering-06.png)
Figure 6. △*M271\_14685/m271\_14690* and NRRL 5491 have produced rapamycin in 11 days.

It can be seen from the above figure that the rapamycin produced by our knockout *△M271\_14685/m271\_14690*
is much higher than that produced by NRRL 5491 (Figure 6). And the amount of rapamycin produced on the ninth
day reached 120mg/L.

## Learn

This indicates that the knockout of the target gene *M271\_14685/M271\_14690* is more conducive to the
secretion of rapamycin by *Streptomyces* *rapamycinicus*. This suggests that the subject is feasible
to improve metabolic pathways by knocking out the two-component system, which can be applied to factories in
the near future.

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# Experiments

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# Results

## I. Construction of double knockout plasmid experiment

### 1. PCR amplification of upstream and downstream homology arms gene of M271\_14685/M271\_14690.

We designed the program by inserting the *M271\_14685/14690* upstream homology arm gene into HindIII and
EcoRI sites of the pKC1139 vector. In order to build our plasmids, we amplified the gene fragments from the
genome of *Streptomyces rapamycinicus* NRRL 5491 by PCR (Figure 1), double-enzyme digestion, and ligase
to pKC1139 carrier.

![](https://static.igem.wiki/teams/4281/wiki/results/t-ecnuas-results-01.jpg)

Figure 1: Gel electrophoresis diagram.

Line 1,3,5 are *M271\_14685/14690* upstream homology arm gene, 1208bp.
Line 2,4,6 are *M271\_14685/14690* downstream homology arm gene, 1144bp.

In Figure 1, a clear and single DNA band at 1kp can be seen, indicating that the upstream and downstream
homology arms of *M271\_14685/M271\_14690* were successfully amplified by PCR.

### 2. Gel electrophoresis of recombinant plasmid pKC-M271\_14685/M271\_14690.

![](https://static.igem.wiki/teams/4281/wiki/results/t-ecnuas-results-02.jpg)

Figure 2:Gel electrophoresis diagram.

Line 1,3,5: recombinant plasmid pKC-M271\_14685/ M271\_14690, 10320bp.
Line 2,4,6: recombinant plasmid pKC-M271\_14685/ M271\_14690 digested with EcoRI/HindIII, 7962 bp, 2358
bp.

After selecting the correct colony by colony PCR, we inoculated it in LB with the antibody and extract the
plasmid. To verify if the plasmid is correct, we did double-enzyme-digestion. It can be seen from the figure
that the size of the plasmid we constructed is correct, and the identification results of double enzyme
digestion are also correct.

### 3. Recombinant plasmid pKC-M271\_14685/ M271\_14690 sequencing analysis.

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 (Figure3-5.), and the plasmid was
successfully constructed.

![](https://static.igem.wiki/teams/4281/wiki/results/t-ecnuas-results-03.jpg)
Figure 3: Global sequence alignment of sequencing.

![](https://static.igem.wiki/teams/4281/wiki/results/t-ecnuas-results-04.jpg)
Figure 4: The details of pKC1139-F sequence alignment.

![](https://static.igem.wiki/teams/4281/wiki/results/t-ecnuas-results-05.jpg)
Figure 5: The details of pKC1139-R sequence alignment.

As shown in the three figures, the sequencing results further demonstrated that the pKC-M271\_14685/
M271\_14690 construction was correct, and consistent with PCR identification results.

### 4. Gel electrophoresis of single cross-over strains.

![](https://static.igem.wiki/teams/4281/wiki/results/t-ecnuas-results-06.jpg)

Figure 6: Gel electrophoresis diagram.

Line 1: single cross-over strain-1, incorrect.
Line 2: single cross-over strain-2, 1575bp, correct.

To construct the engineering strain, we firstly transferred the recombinant plasmid into ET12567/pUZ8002
competent cells and screened the correct strain through 3 antibodies, and cultured it in the liquid medium.
Co-cultured the *E.coli* with *Streptomyces rapamycinicus* and screened for the single cross-over
strain.

We selected two strains to identify the single exchange of gene fragments, and the results showed that the
single cross-over strain 2 was successful (Figure 6).

### 5. Gel electrophoresis of double cross-over strains.

![](https://static.igem.wiki/teams/4281/wiki/results/t-ecnuas-results-07.jpg)

Figure 7: Gel electrophoresis diagram.

Line 1: double cross-over strain,326bp, correct.
Line 2: control strain: *Streptomyces rapamycinicus* NRRL 5491, 3475bp.

Based on the screened single cross-over strain, we further subcultured and screened for the double
cross-over strain, and that’s the engineering strain we needed. We also used colony PCR to verify (Figure
7), and as the figure shows, we construct the strain successfully. Compared with the negative control, the
gene fragments of the engineered bacteria we constructed were successfully exchanged. That is, the
engineered bacteria that knocked out the M271\_14685/M271\_14690 gene were successfully obtained, and we named
the strain *△M271\_14685/m271\_14690*.

## II. Functional test

### 1. HPLC profiles of the wild-type strain *Streptomyces rapamycinicus* NRRL 5491 and the ΔM271\_14685/M271\_14690 mutant.

![](https://static.igem.wiki/teams/4281/wiki/results/t-ecnuas-results-08.jpg)
Figure 8: A is rapamycin, B is wild-type NRRL 5491, and C is
△M271\_14685/m271\_14690.

Co-cultured *Streptomyces rapamycinicus* NRRL 5491 with ΔM271\_14685/M271\_14690 in the fermentation
medium, we collected the sample respectively at 5d,7d,9d, and 11d. We mixed 0.5 mL samples with the same
volume of methanol and tested the yield of rapamycin after centrifuge. To identify the correct peek of
rapamycin, we use the standard rapamycin as positive control. It can be seen from Figure 8 that the
△M271\_14685/m271\_14690 detected more rapamycin at the same time as standard rapamycin.

### 2. Analysis of rapamycin production.

![](https://static.igem.wiki/teams/4281/wiki/results/t-ecnuas-results-09.jpg)
Figure 9: zz △M271\_14685/m271\_14690 and NRRL 5491 have produced rapamycin during 11
days.

It can be seen from the above figure that the rapamycin produced by our knockout △M271\_14685/m271\_14690 is
much higher than that produced by NRRL 5491. And the amount of rapamycin produced on the ninth day reached
120mg/L. This suggests that the subject is feasible to improve metabolic pathways by knocking out the
two-component system, which can be applied for clinical application in the near future.

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