iGEM 2023 — Team izju-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

# iZJU-China

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---

## Engineering

## GLF protein purification

### Design

To verify the successful expression of GLF in the plasmid transferred to Escherichia coli, we purified GLF with
reference to the [standard
protein purification protocol](https://static.igem.wiki/teams/4940/wiki/prokaryotic-protein-expression-and-purification-protocol.pdf).

### Build

Please see [Lab
Notebook](https://static.igem.wiki/teams/4940/wiki/tinywow-lab-notebook-final-37083232.pdf)

### Test

![](https://static.igem.wiki/teams/4940/wiki/engineering/1.jpg)

**Figure 1. GLF expression validation via SDS-PAGE with Coomassie blue staining.** There was
no lanes in E1-E6, which indicates that no protein was successfully purified.CL (Cell lysate), FT (Flow
through), W1-3 (wash 1-3), Protein Marker, E1-6 (elution 1-6).

Failed to extract GLF protein.

### Learn

After completing plasmid transformation, *Escherichia coli* could grow on kanamycin-resistant plates,
indicating that the plasmid was successfully introduced into *Escherichia coli*, but the protein could not
be successfully purified.

Two possible reasons:

1. Plasmid was not expressed after entering *Escherichia coli*.

2. Expressed protein could not be extracted using methods above.

Therefore, we further consulted the relevant information about GLF protein. We found that GLF is a glucose
dislodgment carrier, located on the cell membrane that belongs to the membrane protein. Membrane proteins can be
discarded following cell debris after sonication. This may be one of the important reasons why we failed to
achieve GLF purification.

### Re-design

We therefore looked for ways to purify membrane proteins and finally decided to replace ultrasonic
fragmentation method with triton X-100, which can lyse the cell membrane and thus more fully solubilize membrane
proteins into buffer without discarding them with cell debris ([standard
protein purification protocol](https://static.igem.wiki/teams/4940/wiki/prokaryotic-protein-expression-and-purification-protocol.pdf)).

### Re-build

Please see [Lab
Notebook](https://static.igem.wiki/teams/4940/wiki/tinywow-lab-notebook-final-37083232.pdf)

### Re-test

![](https://static.igem.wiki/teams/4940/wiki/engineering/2.jpg)

**Figure 2. GLF expression validation via SDS-PAGE with Coomassie blue staining.**  The
results suggest that the expressions of glf were successfully detected, which was indicated by the corresponding
bands around 50kDa, 53kDa, 28kDa and 25kDa. CL (Cell lysate), FT (Flow through), W1-3 (wash 1-3), Protein
Marker, E1-6 (elution 1-6).

Red border marked in the figure: successfully purified 50kDa target protein GLF.

### Learn

Our previous learning about GLF is correct. Since GLF is a membrane protein, the ultrasonic fragmentation
method cannot effectively separate target protein from cells. The triton X-100 method can successfully extract
GLF. But we also extracted lots of other proteins, which are probably membrane proteins with his-tag removed
from cell membrane by triton X-100.

## Aqueous-organic two-phase system

### Design

Since α-pinene oxide is toxic and insoluble in inorganic solutions, but the degradation of α-pinene by bacteria
introduced into the prα-pol plasmid needs to be carried out in a way that fully maintains the activity of the
cells, the bacteria need to live in LB medium to complete the degradation. How to dissolve organic α-pinene
oxide in inorganic LB medium is a problem that requires special consideration. Therefore, we designed a biphasic
device in which the organic and inorganic phases were placed in a conical flask and shaken by a shaker to mix
them well. It allows the engineering bacteria in the inorganic phase to have full access to the α-pinene oxide
in the organic phase for efficient degradation, while avoiding bacterial death due to continuous contact between
the engineering bacteria and α-pinene oxide.

### Build

Please see [Lab
Notebook](https://static.igem.wiki/teams/4940/wiki/tinywow-lab-notebook-final-37083232.pdf)

### Test

Our Erlenmeyer flask containing aqueous-organic two-phase system was placed in a communal shaker. However,
α-pinene oxide continued to leak through the pore of the sealing film. Experiment was aborted.

### Learn

α-pinene oxide is volatile, which is accelerated by shaking and heating, but normal life of E. coli requires a
constant supply of oxygen, and we could not completely seal the container., We chose to use the decolorization
shaker after communicating with PIs to solve this problem. The decolorization shaker was placed in the fume
cupboard, so that the escaping α-pinene oxide would be absorbed without affecting experimental safety.

### Re-design

We adjusted the aqueous-organic two-phase system as follows:

Adding 4ml of bacterial solution containing Escherichia coli with the introduced prα-pol plasmid and 1ml of
pure α-pinene oxide to the culture tubes.

![](https://static.igem.wiki/teams/4940/wiki/wikipic/finalversion/safety/culture.jpg)

**Figure 3. The structure of simplified aqueous-organic two-phase system.** The components
of the device are indicated in the figure.

### Re-build

Please see [Lab
Notebook](https://static.igem.wiki/teams/4940/wiki/tinywow-lab-notebook-final-37083232.pdf)

### Re-test

In this device, the organic and inorganic phases can be fully mixed and play the same mixing role as that of
the thermostatic shaker, but it is impossible to maintain the temperature at 37℃. Therefore, we chose to
incubate the bacteria in the thermostatic shaker until the OD value reaches about 0.5, and then take it out and
add it into the culture tube to incubate with shaking for 3.5h.

### Learn

This incubation was successful and we removed the organic phase after 3.5h and sent it to the Chemical Analysis
Centre for GC-MS, where α-pinene oxide was finally detected.

![](https://static.igem.wiki/teams/4940/wiki/engineering/4.png)

**Figure 4. The mass spectrum was generated for analysis.** This revealed that both
substrates, α-pinene oxides **(A)**, and product,isonovaval **(B)** were detected in the GC-MS analysis.

![](https://static.igem.wiki/teams/4940/wiki/engineering/5.png)

**Figure 5. TIC spectrum of four groups.** During The ion abundance was the highest in the
IPTG group **(A)** compared to that in the group without IPTG induction **(D)** during 6-8 min and 13-16
min. However, the ion abundance was similar in the PBS group **(C)** and pure α-pinene oxides group
**(B)**, both of which were lower than the other groups.

37℃ is the optimal temperature for the degradation of α-pinene oxide by E. coli introduced into the prα-pol
plasmid, there is no way to keep the temperature at 37℃ using this device, so it will have a certain impact on
the survival of the engineered bacteria and the degradation process, and subsequently, we consider the use of a
constant temperature shaker to optimise the degradation environment.

## Co-transformation

### Design

In order to express both GLF plasmid and P450-GlcDH plasmid in the same *E. coli*, we referred to the
protocol for chemical transformation (add a hyperlink to the protocol for chemical transformation) and added
half of the total plasmid amount of GLF plasmid and P450-GlcDH plasmid, respectively, and carried out chemical
transformation.

### Build

Please see [Lab
Notebook](https://static.igem.wiki/teams/4940/wiki/tinywow-lab-notebook-final-37083232.pdf)

### Test

![](https://static.igem.wiki/teams/4940/wiki/engineering/2023-10-12-22-05-19.png)

**Figure 6. Colony growth on the plate after chemical cotransformation.** We transferred GLF
plasmid and P450-GlcDH plasmid into BL21 and took 200μL respectively on LB, kanamycin resistant, streptomycin
resistant and dual-resistant plates one hour after resuscitation. **a.** Non-resistant (LB) plates have
colony growth. **b.** Kanamycin resistant plates had colony growth. **c.** Streptomycin resistant plates
have colony growth. **d.** Double resistance (kanamycin resistance and streptomycin resistance) on the plate
without colony growth.

### Learn

Considering the inefficiency of chemical transformation, which may be responsible for the fact that there is no
way for two plasmids to enter a bacterium at the same time, we plan to use the more efficient electroshock
transformation.

### Re-design

We referred to the protocol for [electrotransformation](https://static.igem.wiki/teams/4940/wiki/electrotransformation-protocol.pdf)
and added half of the total plasmid amount of GLF plasmid and P450-GlcDH plasmid, respectively, for
electrotransformation.

### Re-build

Please see [Lab
Notebook](https://static.igem.wiki/teams/4940/wiki/tinywow-lab-notebook-final-37083232.pdf)

### Re-test

![](https://static.igem.wiki/teams/4940/wiki/engineering/7.png)

**Figure 7. Growth of bacterial colonies on the plate after electroporation of E. coli.** A
total of four groups of electroporation (1) P450-GlcDH plasmid (2) GLF plasmid (3)(4) P450-GlcDH plasmid and GLF
plasmid were transformed into E.coli BL21, respectively. After electroporation, the bacteria were inoculated
into liquid LB medium and shaken in a constant temperature incubator for one hour. (1) (2) (3) After
centrifugation, 800μL supernatant was discarded, leaving 200μL suspensible bacteria. (1) was inoculated on a
streptomycin resistant plate **(a)**, (2) was inoculated on a kanamycin resistant plate, and (3) was
inoculated on a streptomycin, kanamycin double resistant plate **(b)**. (4) After centrifugation, the 200μL
supernatant was discarded, leaving 800μL heavy suspension bacteria, and divided into four parts, respectively,
on **(d)** non-resistant LB plate, **(e)** streptomycin resistant plate, **(f)** kanamycin resistant
plate, and **(g)** double resistant plate. All plates were cultured in a constant temperature incubator at
37°C for 12h.

Colonies grew only when BL21 E. coli introduced with the glf single plasmid was applied to kanamycin-resistant
plates, and when BL21 E. coli introduced with the GLF plasmid and the P450-GlcDH plasmid was applied to
non-resistant plates, suggesting that electrotransformation also did not enable the two plasmids to be
introduced into a single bacterium at the same time.

### Learn

According to the results, the P450 plasmid did not manage to enter the bacteria even during the
electrotransformation of a single plasmid, so we considered using the chemical transformation method first by
introducing the P450 plasmid into BL21 E. coli, then restoring the sensory state of these E. coli, and then
transfecting the glf plasmid into the same tube of BL21 E. coli by electrotransformation.

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GLF protein purification

Aqueous-organic two-phase system
Co-transformation


# iZJU-China

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---

## Results

## Result & Discussion

Our group takes a long time to accomplish the whole process. What we have performed is shown below. Detailed
information including experimental design and protocols are described on the Experiment page.

### We want to:

Oxidize α-pinene efficiently by P450BM-3 QM combined with NADH regenerative system, which is constructed by a
membrane-bound protein GLF and intracellular enzyme GlcDH-II.
Degrade α-pinene oxide through an enzyme, Prα-POL from Pseudomonas rhodesiensis CIP107491.

![](https://static.igem.wiki/teams/4940/wiki/wikipic/finalversion/results/result-figure1.png)

**Figure 1. The process of the whole project, starting from engineered
bacteria construction to the determination of the degradation efficiency.**

### How did we do it?

#### Gibson assembly

![](https://static.igem.wiki/teams/4940/wiki/wikipic/finalversion/results/result-figure2.png)

**Figure 2. The general principles of Gibson assembly.** This
depends on the recombination of the overlap between the target gene and the vector. The result of transformation
is checked by the LB agar plate with corresponding antibiotics.

#### Competent cell transformation

Three recombinant plasmids were transformed E. coli BL21 star (DE3) for gene expression respectively. The
colonies shown on the agar plates containing antibiotics indicated that the three plasmids were all successfully
transformed into E. coli BL21 star (DE3) (Figure 3).

![](https://static.igem.wiki/teams/4940/wiki/wikipic/finalversion/results/figure-3-a-b-c.png)

**Figure 3. Transformation results of three plasmids in the LB agar
plate**. Three plasmids were all successfully transformed into bacteria, indicated by the colonies on the
agar plate. (A) E. coli BL21 star (DE3) with pQE-80L-Kan glf; (B) E. coli BL21 star (DE3) with pCDFDUET-1
p450bm-3qm glcdh-II; (C) E. coli BL21 star (DE3) pET-28a prα-pol.

#### Prokaryotic protein expression

To induce protein expression, IPTG was added into the bacteria culture when OD600 equaled 0.5-0.6,
approximately 4 h after inoculation and then cultured for another 4 h (Figure 4).

![](https://static.igem.wiki/teams/4940/wiki/wikipic/finalversion/results/figure-4.png)

**Figure 4. The general pattern of E. coli growth.** The change of
OD600 of the bacterial culture along with time was shown. OD600 was recorded at 0, 1, 2, 3 and 4 h, and IPTG was
added at 4 h.

#### Protein purification and SDS-PAGE

The proteins are all conjugated to a 6xhis tag, which can be purified by His-tag Protein Purification Kit. We
can observe the target bands through SDS-PAGE with Coomassie brilliant blue staining.

![](https://static.igem.wiki/teams/4940/wiki/wikipic/finalversion/results/figure-5-1.png)
![](https://static.igem.wiki/teams/4940/wiki/wikipic/finalversion/results/figure-5-2.png)
![](https://static.igem.wiki/teams/4940/wiki/wikipic/finalversion/results/figure-5-3.png)

**Figure 5. Glf, P450BM-3 QM, GLcDH-II, Prα-POL expression
validation via SDS-PAGE with Coomassie blue staining.** The results suggest that the expressions of glf,
P450BM-3 QM, GLcDH-II, Prα-POL were successfully detected, which was indicated by the corresponding bands around
50kDa, 53kDa, 28kDa and 25kDa. However, the bands of protein GLF were relatively blurry in all lanes.
CL (Cell lysate), FT (Flow through), W1-3 (wash 1-3), Protein Marker, E1-6 (elution 1-6).

#### Detection of engineered bacterial susceptibility to α-pinene oxides

To better determine the concentration of α-pinene oxides used in aqueous-organic two-phase system, corn oil was
used as solvent to dilute α-pinene oxides. Kirby-Bauer test was used to detect the engineered bacterial
sensitivity to α-pinene oxides. The results revealed that E. coli BL21 star (DE3) pET-28a prα-pol showed
low-sensitivity to α-pinene oxides.

![](https://static.igem.wiki/teams/4940/wiki/wikipic/finalversion/results/figure-6.png)

**Figure 6. Results of preliminary experiment. (A)** .The two
centrifugal tubes originally contained 1 ml corn oil, and 0.8 ml α-pinene oxides was added twice each time into
the left tubes. No stratification was observed. **(B)**Kirby-Bauer test. A total of three groups of repeats
were performed. 75% and 100% concentrations (v/v) of α-pinene oxides dissolved in corn oil were used.
Antibacterial rings were less than 3 mm in all groups, and the 75% and 100% experimental groups had similar
antibacterial rings in each repeat group.

#### Aqueous-organic two-phase system with GC-MS analysis

In order to test whether the engineered bacteria can degrade the target substances efficiently, an
aqueous-organic two-phase system was constructed. The mass spectrum showed that both substrates, α-pinene
oxides, and product, isonovaval were detected. In addition, an obvious difference can be seen between 6-8 min
and 13-16 min in the Total ion chromatogram (TIC) of the IPTG group. This suggests that the certain reaction
that converts α-pinene oxides to isonovaval may appear in the engineered bacteria.

![](https://static.igem.wiki/teams/4940/wiki/wikipic/finalversion/results/figure-7-a-b.png)

**Figure 7. The mass spectrum was generated for analysis.**This
revealed that both substrate, α-pinene oxides**(A)**, and product, isonovaval **(B)** were detected in the
GC-MS analysis.

![](https://static.igem.wiki/teams/4940/wiki/wikipic/finalversion/results/figure-8-a-b-c-d.png)

**Figure 8. TIC spectrum of four groups.** During The ion
abundance was the highest in the IPTG group **(A)**compared to that in the group without IPTG induction
**(D)** during 6-8 min and 13-16 min. However, the ion abundance was similar in the PBS group **(C)** and
pure α-pinene oxides group **(B)** both of which were lower than the other groups.

### Discussion

We have successfully cloned and expressed three plasmids into E. coli BL21 star (DE3) and validated the
expression of each protein. However, the bands of GLF were not as clear as other bands, which can be due to the
properties of GLF protein. GLF is an insoluble membrane-bound protein and may also be expressed at a low level,
thus hard to be purified by our protocols. In addition, further analysis is required to determine the
degradation efficiency of both engineered bacteria.

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We want to:
How did we do it?
Gibson assembly
Competent cell transformation
Prokaryotic protein expression
Protein purification and SDS-PAGE
Detection of engineered bacterial susceptibility to α-pinene oxides
Aqueous-organic two-phase system with GC-MS analysis
Discussion