iGEM 2022 — Team fudan

{% extends "layout.html" %}
{% block page\_author %}Yujian SONG{% endblock %}
{% 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 side\_bar %}

* [January](#january)
* [February](#february)
* [March](#march)
* [April](#april)
* [May](#may)
* [June](#june)
* [July](#july)
* [August](#august)
* [September](#september)
* [October](#october)

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{% block page\_content %}

## January 2022

Wet Lab: Zhongyi Yang, Weiwen Chen.

Dry Lab: Yunjia Liu.

HP: Weiyi Li, Zihan Wang, Yihan Fang.

![](https://static.igem.wiki/teams/4162/wiki/team/notebook/2022-1.jpg)

## February 2022

Wet Lab: Zhongyi Yang, Weiwen Chen.

Dry Lab: Yunjia Liu.

HP: Weiyi Li, Zihan Wang, Yihan Fang.

## March 2022

Wet Lab: Zhongyi Yang, Weiwen Chen.

Dry Lab: Yunjia Liu, Zhiyue Chen.

HP: Weiyi Li, Zihan Wang, Yihan Fang.

### April 2022

Wet Lab: Lock down. No lab access.

Dry Lab: Zhiyue Chen, Yunjia Liu.

HP: Lock down. No outreach activities.

### May 2022

Wet Lab: Lock down. No lab access.

Dry Lab: Zhiyue Chen, Yunjia Liu.

HP: Lock down. No outreach activities.

### June 2022

Wet Lab: Lock down and no lab access until August.

Dry Lab: Zhiyue Chen, Yunjia Liu.

HP: Weiyi Li, Zihan Wang；Yihan Fang.

### July 2022

Wet Lab: Lock down and no lab access until August.

Dry Lab: Zhiyue Chen, Yunjia Liu.

HP: Weiyi Li, Zihan Wang；Yihan Fang.

![](https://static.igem.wiki/teams/4162/wiki/team/notebook/2022-7.jpg)

### August 2022

Wet Lab: Zhongyi Yang, Weiwen Chen, Yujian Song.

Dry Lab: Zhiyue Chen, Yunjia Liu.

HP: Weiyi Li, Zihan Wang；Yihan Fang.

![](https://static.igem.wiki/teams/4162/wiki/team/notebook/2022-8.jpg)

### September 2022

Wet Lab: Zhongyi Yang, Weiwen Chen, Yujian Song.

Dry Lab: Zhiyue Chen, Yunjia Liu.

HP: Weiyi Li, Zihan Wang；Yihan Fang.

![](https://static.igem.wiki/teams/4162/wiki/team/notebook/2022-9a.jpg)
![](https://static.igem.wiki/teams/4162/wiki/team/notebook/2022-9b.jpg)

### October 2022

Wet Lab: Zhongyi Yang, Weiwen Chen, Yujian Song.
Check [here](https://static.igem.wiki/teams/4162/wiki/notebook-wetlab1.pdf) for additional records.

Dry Lab: Zhiyue Chen, Yunjia Liu.

HP: Weiyi Li, Zihan Wang；Yihan Fang.

![](https://static.igem.wiki/teams/4162/wiki/team/notebook/2022-10a.jpg)
![](https://static.igem.wiki/teams/4162/wiki/team/notebook/2022-10b.png)
![](https://static.igem.wiki/teams/4162/wiki/team/notebook/2022-10c.png)

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{% extends "layout.html" %}
{% block page\_author %}Weiwen CHEN{% endblock %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block side\_bar %}

* [Polycistronic expression](#ribozyme-assisted-polycistronic-co-expression-system-prap)

  ![](https://static.igem.wiki/teams/4162/wiki/arrow-orange.png)

  + [Polycistronic expression](#ribozyme-assisted-polycistronic-co-expression-system-prap)
  + [Cycle 1](#cycle-1)
  + [Cycle 2](#cycle-2)
* [HPLC for product validation](#application-of-hplc-in-product-detection)

  ![](https://static.igem.wiki/teams/4162/wiki/arrow-orange.png)

  + [HPLC for product validation](#application-of-hplc-in-product-detection)
  + [Cycle 1](#cycle-1-1)
  + [Cycle 2](#cycle-2-1)
* [References](#references)

{% endblock %}
{% block page\_content %}

In the process of building single-cell factory, we encountered several problems in [parts](/fudan/parts) design and experiments, and tried to solve them following iterations of the **DBTL** cycle (Design, Build, Test, Learn).

## Ribozyme-assisted polycistronic co-expression

### Cycle 1

#### Design

As many researches indicate, the major problem of polycistronic vectors, which contain two or more target genes under one promoter, is the much lower expression of the downstream genes compared with that of the first gene next to the promoter (Kim, Kyung-Jin et al. 2004). The tail of the coding sequence (CDS) can interfere with the head of the ribosome binding site (RBS), which can hinder RBS from combining to ribosomes. Such shortage occurred when we assembled crtEBIY sequentially only to find incomplete expressions of our target proteins.

To optimize the crt BioBrick, we adopted hammerhead ribozyme [BBa\_K4162005](http://parts.igem.org/Part%3ABBa_K4162005) to construct a ribozyme-assisted polycistronic co-expression BioBricks. Through inserting ribozyme sequences between crtEBIY, the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons which have comparable translation efficiency. By building this system, we expected that the self-interaction of the polycistron can be avoid.

#### Build

We first assemble four basic modules: [BBa K4162010](http://parts.igem.org/Part%3ABBa_K4162010) (ribozyme + T7\_RBS + crtE), [BBa\_K4162013](http://parts.igem.org/Part%3ABBa_K4162013) (ribozyme + T7\_RBS + crtB), [BBa\_K4162016](http://parts.igem.org/Part%3ABBa_K4162016) (ribozyme + T7\_RBS + crtI) and [BBa\_K4162019](http://parts.igem.org/Part%3ABBa_K4162019) (ribozyme + T7\_RBS + crtY). Next, we apply overlapping PCR to assemble 2-3 random basic modules. Finally, we obtained several BioBricks composed of four basic modules assembled in different orders.

![crt-biobrick.png (5827×1260) (igem.wiki)](https://static.igem.wiki/teams/4162/wiki/cww/crt-biobrick.png)

**Figure 1.** Module crt IYEB [BBa\_K4162021](http://parts.igem.org/Part%3ABBa_K4162021), an example of composite modules which contain four basic modules.

#### Test

We tested the protein expressions of different combinations of random carotene synthesis enzymes through SDS-PAGE, and as Figure 2 and 3 indicate, each enzyme can be expressed successfully.

![](https://static.igem.wiki/teams/4162/wiki/cww/3e.png)

**Figure 2.** IPTG(-/+) = without/with 0.2 mM IPTG for 3-6 hours, adding IPTG to a bacteria culture with OD600 0.2-0.3. M: Protein molecular weight marker ladder. Lane 1~4, 6~9, 11~17: pET28 plasmids encoding crtYB, IB, EY separated by self-cleaving ribozyme, crtB, crtE, crtY without any tag were transformed into BL21(DE3) HI-Control strain, single clones (1YB1/IB7/IB8/1B1/EY3/EY4/1E1/1Y1) were picked for liquid LB culture. Lane 10: pET28 plasmids encoding crtI were transformed into BL21(DE3) HI-Control strain, single clones (1I7) were picked for liquid LB culture. Protein expression was induced in parallel cultures by IPTG. Bacterial cultures were monitored by OD600, and 5x10^7 cells were harvested by centrifugation and lysis in 1x SDS sample buffer. Equal amount (10 μL, 2x10^6 cells) of whole cell lysate were analyzed by SDS-PAGE (4~20% gradient gel, Tanon brand). Red arrows point to crtI protein. Green arrows point to crtY protein. Black arrows point to crtB protein. Yellow arrows point to crtE protein.

![](https://static.igem.wiki/teams/4162/wiki/cww/3f.png)

**Figure 3.** IPTG(-/+) = without/with 0.2 mM IPTG for 3-6 hours, adding IPTG to a bacteria culture with OD600 0.2-0.3. M: Protein molecular weight marker ladder. Lane 4~16: pET28 plasmids encoding crtBEI separated by self-cleaving ribozyme, crtI, crtEI, crtB, crtEB, crtIYEB separated by self-cleaving ribozyme without any tag were transformed into BL21(DE3) HI-Control strain, single clones (BEI/1I7/1EI/1B1/EB5/EB6/IYEBa) were picked for liquid LB culture. Lane 1~2: pET28 plasmids encoding BCMO-ybbO-tdMCP-EGFP were transformed into BL21(DE3) HI-Control strain, single clones (2BY14tG14) were picked for liquid LB culture. Lane 3~4: pET28 plasmids encoding BCMO-ybbO were transformed into BL21(DE3) HI-Control strain, single clones (BY14hi) were picked for liquid LB culture. Protein expression was induced in parallel cultures by IPTG. Bacterial cultures were monitored by OD600, and 5x10^7 cells were harvested by centrifugation and lysis in 1x SDS sample buffer. Equal amount (10 μL, 2x10^6 cells) of whole cell lysate were analyzed by SDS-PAGE (4~20% gradient gel, Tanon brand). Red arrows point to crtI protein. Green arrows point to crtY protein. Black arrows point to crtB protein. Yellow arrows point to crtE protein.

#### Learn

We constructed successfully enabled the expression of each enzyme contained in the crt polycistron in comparable proportions. However, if one wants to precisely manage the proportion of expression products of individual CDSs in this polycistron, it is not enough to separate the CDSs by ribozyme sequences. Besides, the stablility of expression among clones cannot be confirmed by electrophoresis experiments.

### Cycle 2

#### Design

Next, we designed composite modules adjusted for the RBS of individual enzymes and performed incubation and centrifugation experiments to probe the stability of expression.

#### Build

Typical modules we designed are [BBa\_K4162117](http://parts.igem.org/Part%3ABBa_K4162117) and [BBa\_K4162118](http://parts.igem.org/Part%3ABBa_K4162118).

BBa\_K4162117: This BioBrick was created through overlapping PCR of [BBa\_K4162020](http://parts.igem.org/Part%3ABBa_K4162020) (ribozyme+J6\_RBS+crtY), [BBa\_K4162010](http://parts.igem.org/Part%3ABBa_K4162010) (ribozyme+T7\_RBS+crtE), [BBa\_K4162013](http://parts.igem.org/Part%3ABBa_K4162013) (ribozyme+T7\_RBS+crtB) and [BBa\_K4162016](http://parts.igem.org/Part%3ABBa_K4162016) (ribozyme+T7\_RBS+crtI). In this part, the RBS of crtEBI has equal intensity while the RBS of crtY is significantly weaker than the others. Because crtY catalyzes the last step of the carotenoid reaction chain, we guess the concentration of substrate catalyzed by this enzyme is significantly lower than for the first three steps of the reaction. To avoid the problem of flux imbalance in biosynthesis as well as to reduce unnecessary metabolic stress on cells, we intentionally weakened the RBS intensity of crtY.

![](https://static.igem.wiki/teams/4162/wiki/cww/yebi.png)

**Figure 4.** Module crtYEBI, using a [weak](http://parts.igem.org/Part%3ABBa_J61100) RBS drives crtY expression.

BBa\_K4162118: This BioBrick was created through overlapping PCR of [BBa\_K4162020](http://parts.igem.org/Part%3ABBa_K4162020) (ribozyme+J6\_RBS+crtY), [BBa\_K4162010](http://parts.igem.org/Part%3ABBa_K4162010) (ribozyme+T7\_RBS+crtE), [BBa\_K4162013](http://parts.igem.org/Part%3ABBa_K4162013) (ribozyme+T7\_RBS+crtB) and [BBa\_K4162016](http://parts.igem.org/Part%3ABBa_K4162016) (ribozyme+T7\_RBS+crtI). In this part, the RBS of crtBIY has equal intensity while the RBS of crtE is significantly stronger than the others. Since crtE catalyzes the first step of the carotenoid reaction chain, increase the concentration of product catalyzed by this enzyme is beneficial for the remaining three steps. To avoid more serious flux imbalance problems, we boosted the RBS intensity of crtE only in this BioBrick and explored whether the carotenoid production of the strain could be significantly enhanced.

![](https://static.igem.wiki/teams/4162/wiki/cww/ebiy.png)

**Figure 5.** Module crtEBIY, using a [strong](http://parts.igem.org/Part%3ABBa_B0030) RBS drives crtE expression.

#### Test

We transformed plasmids into *E. coli* DH5α and cultured it, and finally obtained YELLOW bacterial pellet after centrifugation.

Figures 6 to 8 show that transformed *E. coli* successfully expressed the target enzyme and yielded β-carotene. In Figure 6, it can be seen that module YEBI corresponds to a darker orange color of the post-centrifugation precipitation compared to module YBEI ([BBa\_K4162119](http://parts.igem.org/Part%3ABBa_K4162119)), characterizing the superior carotenoid yielding ability of module YEBI. As shown in Figure 8, different clones of *E. coli* transformed with the same plasmid were cultured using 96-well plates. After shaking culture overnight, the plate was spin at 4500 rpm for 30 minutes and bacteria was pelleted to the bottom of the wells. There is no clonal variation, showing stable expression of the individual mono-cistrons separated by ribozyme sequences (i.e., no trucated expression nor null function protein of downstream genes due to being placed under one promoter).

![117-119.jpg (3840×2160) (igem.wiki)](https://static.igem.wiki/teams/4162/wiki/cww/117-119.jpg)

**Figure 6.** The centrifuge tubes containing module [crtYEBI](http://parts.igem.org/Part%3ABBa_K4162119) (first from the left) and module crtYBEI [BBa\_K4162119](http://parts.igem.org/Part%3ABBa_K4162119) (second from the left) contain visible yellow bacterial pellet.

![118.jpg (2160×3840) (igem.wiki)](https://static.igem.wiki/teams/4162/wiki/cww/118.jpg)

**Figure 7.** Overnight culture of bacterial expressing module [crtEBIY](http://parts.igem.org/Part%3ABBa_K4162118) was centrifuged at 13000 rpm for 1 minute.

![96a-117.jpg (3840×2160) (igem.wiki)](https://static.igem.wiki/teams/4162/wiki/cww/96a-117.jpg)

**Figure 8.** **96-well plate of module [crtYEBI](http://parts.igem.org/Part%3ABBa_K4162117).** Except for the blank control well marked in black, all clones growing different wells had similar β-carotene content in the bacterial pellet.

#### Learn

Multiple cis-trans substrates assembled using nucleases have excellent stablility of expression among clones. And we saw the possibility to manage individual cistron by adjusting the stength of its upstream RBS.

## Apply HPLC to detect β-carotene and retinol

### Cycle 1

#### Design

Prior to 2022 Team Fudan, teams aiming to synthesize β-carotene conducted visual test to determine carotenoid production by looking at shades of orange. After we observed the color change of the system, for quantitative detection and validation with chemical standards, we applied HPLC to detect β-carotene and retinoids.

#### Build

After searching for related information, we initially decided to adopt the following protocol:
Chromatographic column: C18; mobile phase: carotene methanol:acetonitrile 70:30; retinoids methanol:acetonitrile 95:5; flow rate: 1.5 mL/min; column temperature: 40 degrees celsius; wavelength: retinal 370 nm, retinol 340 nm, carotene 454 nm (Yoon, Sang-Hwal et al. 2009).

#### Test

In the first few rounds of experiments, the absorption peaks of carotenoids were obvious but the absorption peaks of retinol and retinal could not be obtained.

#### Learn

We should continue to consult with professionals.

### Cycle 2

#### Design

We modified the protocol according to the instrument.

#### Build

New protocol: Agilent liquid chromatograph (HPLC-DAD); column C18 (250mm); column temperature 30°C; mobile phase methanol:water = 96:4; flow rate 0.8 mL/min; detection wavelength 325nm.
The protocol is from [Boulder Research (Guangzhou) Biotechnology Co](https://mp.weixin.qq.com/s/81zY_LoMratSEOOy7JUaFw).

### Test

![beta.png (1071×336) (igem.wiki)](https://static.igem.wiki/teams/4162/wiki/cww/beta.png)

**Figure 9.** 1:10 dilution of methanolic saturated solution of β-carotene.

![3-7.png (1075×353) (igem.wiki)](https://static.igem.wiki/teams/4162/wiki/cww/3-7.png)

![3td7.png (1075×362) (igem.wiki)](https://static.igem.wiki/teams/4162/wiki/cww/3td7.png)

**Figure 10.** Acetone extracted samples were subjected to HPLC (C18 column)as described previously. The extract from bacteria expressing CAG-MS2, BCMO, ybbO, crtYEBI (the panel above, short name 3-7) compared with the extract from bacteria expressing CAG-MS2, BCMO, ybbO-tdMCP, crtYEBI (the panel below, short name 3td7), there are two peaks in the figure between 2-4 minutes (where our standards appear [as well others](https://mp.weixin.qq.com/s/81zY_LoMratSEOOy7JUaFw), detected at 325 nm), the absorbance value of the two peaks of 3td7 in this period is significantly higher than that of 3-7.

### Learn

Clear results can be obtained for the amount of β-carotene converted. Without a standard, the appropriate chromatographic separation and identification of retinol remains to challenging.

## References

Kim, K. J., Kim, H. E., Lee, K. H., Han, W., Yi, M. J., Jeong, J., & Oh, B. H. (2004). Two-promoter vector is highly efficient for overproduction of protein complexes. Protein science : a publication of the Protein Society, 13(6), 1698–1703.

Yoon, S. H., Lee, S. H., Das, A., Ryu, H. K., Jang, H. J., Kim, J. Y., Oh, D. K., Keasling, J. D., & Kim, S. W. (2009). Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli. *Journal of biotechnology*, *140*(3-4), 218–226.

{% endblock %}

{% extends "layout.html" %}
{% block page\_author %}Zhongyi YANG{% endblock %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block side\_bar %}

* [Cryopreservation](#cell-cryopreservation)
* [Recovery](#cell-recovery)
* [Colony PCR](#colony-pcr)
* [OE PCR](#overlap-extension-pcr-oe-pcr)
* [Agarose Gel](#agarose-gel-electrophoresis)
* [Transformation](#plasmid-transformation)
* [Miniprep](#plasmid-miniprep)
* [Lysate for SDS-PAGE](#preparation-of-bacterial-cell-lysate-for-sds-page)
* [SDS-PAGE](#sds-page)
* [IPTG Induction](#iptg-induction-experiment)
* [Agarose Pad for Microscopy](#preparation-of-agarose-pad-for-microscopy)
* [Acetone Extraction](#acetone-extraction-of-bacteria-pellet-for-hplc)

{% endblock %}
{% block page\_content %}

## Bacteria Cryopreservation

Cryopreservation is a technique that stores cells at a very low temperature (-80℃) to reduce cell metabolic damage and enable long-term storage.

1. Add cryoprotectant: Add 80% glycerin 500 μL and bacterial fluid 1000 μL into Cryopreservation vials. Mix upside down gently.
2. Seal the vials.
3. Store the vials: Store prepared vials in the refrigerator at -80℃.

## Bacteria Recovery

Bacteria preserved at -80℃ need to be recovered to restore cell growth.

1. Prepare plate: Take out the plate with relevant resistance from 4℃ and wait for the temperature to the room temperature. Mark at the bottom of the plate.
2. Recover the cell: The -80℃ frozen strain was taken out and placed on the ice quickly. Take a ring of the upper layer of melted bacteria liquid with a sterilized inoculation ring. Open the cap and draw a line on the plate.
3. Culture the recovered cell: Incubate at 37℃ overnight.

## Colony PCR

PCR is performed to amplify DNA fragments in our project. We have publish [it on protocols.io](https://www.protocols.io/view/colony-pcr-for-2022-igem-rm7vzb352vx1/v1).

1. Select colonies on the agar plate to pick. Culture portion of the colony in LB media with antibiotic and grow at 37℃. Put the remainder of the colony into a correspondingly numbered PCR tube with 20 µL of reaction mix (as below), and pipetting to complete resuspend the bacteria.

   | Reagent | Volume/μL |
   | --- | --- |
   | 10x Taq Buffer | 2 |
   | 10 μM Forward Primer | 0.5 |
   | 10 μM Reverse Primer | 0.5 |
   | Template DNA (bacteria colony) | added later |
   | dNTP 10mM mix | 0.5 |
   | Taq DNA polymerase | 0.5 |
   | distilled water | 16 |
   | Total | 20 |
2. Prepare PCR reaction mix before step 1, and place the mix on ice.

   Primers used for screening crt genes insertion are:
   > 5-crtB
   5-ATGAATAATCCGTCGTTACTCAATCATGC-3
   > 5-crtE
   5-ATGACGGTCTGCGCAAAAAAAC-3
   > 5-crtI
   5-ATGAAACCAACTACGGTAATTGGTGC-3
   > 5-crtY
   5-ATGCAACCGCATTATGATCTGATTC-3
   > rev320crtB
   5-CCTTCCAGATGATCAAACGCGTAAG-3
   > rev320crtE
   5-ATGAGAATGAATGGTAGGGCGTC-3
   > rev320crtI
   5-GGATTAAACTGCTGAATCTGCGCTTC-3
   > rev320crtY
   5-CCGCGGTATCCATCCACAAG-3

   | Temperature | Time |
   | --- | --- |
   | 94℃ | 05:00 |
   | 94℃ | 00:30 |
   | 60℃ | 00:30 |
   | 72℃ | 03:30, cycle 25 times |
   | 72℃ | 05:00 |
   | 16℃ | ∞ |
3. The PCR program above is for screening crt genes insertion this year.
4. Agarose gel electrophoresis of PCR products. One lane on the gel is loaded with 6 μL D2000 DNA ladder. Run the gel at 100 V for 20-30 minutes, and examine the gel under UV.
5. The correct bacterial clones are sent for Sanger sequencing. Once verified, these clones would be used for further experiments.

## Overlap Extension PCR (OE PCR)

OE PCR is used to fuse fragments.

1. First-round PCR: Use primer a/b to amplify fragment AB by PCR, and c/d to amplify fragment CD. Set up PCR system:

   | Reagent | Volume/μL |
   | --- | --- |
   | distilled water | To 25 |
   | 2 x Phanta Max Buffer | 12.5 |
   | dNTP | 0.5 |
   | Phanta DNA polymerase | 0.5 |
   | DNA Template | Plasmid: 0.5 PCR product: 1 |
   | Forward Primer | 1 |
   | Reverse Primer | 1 |
2. Set up reaction program:

   | Procedure | Temperature | Time | Cycle |
   | --- | --- | --- | --- |
   | Initialization | 95℃ | 30s | 1 |
   | Denaturation | 95℃ | 15s | 30 |
   | Annealing | depends on the primer | 15s |  |
   | Extension | 72℃ | 30s/kb |  |
   | Final elongation | 72℃ | 5min | 1 |
3. Annealing of homologous regions in Second round PCR: Separate and purify fragment AB and CD by agarose gel electrophoresis and DNA gel extraction. Measure the concentration of the two fragments. Dilute them into 1:1 as a template. Set up PCR system:

   | Reagent | Volume |
   | --- | --- |
   | distilled water | To 50 μL |
   | 2 x Phanta Max Buffer | 25 μL |
   | dNTP | 1 μL |
   | Phanta | 1 μL |
   | Template | 0.08 pmol + 0.08 pmol |
4. Set up reaction program:

   | Procedure | Temperature | Time | Cycle |
   | --- | --- | --- | --- |
   | Initialization | 94℃ | 5min | 1 |
   | Denaturation | 94℃ | 30s | 12 |
   | Annealing | 60/64/68/72℃ | 30s |  |
   | Extension | 72℃ | 1min/kb |  |
   | Final elongation | 72℃ | 7min | 1 |
5. Check the products by agarose gel electrophoresis.

## Agarose Gel Electrophoresis

Agarose gel electrophoresis is performed to separate and confirm whether our PCR were correct.

1. Make gel solution: Add 0.7 g agarose, 70 mL TAE buffer into a glass bottle and heat in a micro-oven for 2 min. Cool the liquid agarose gel to lower than 60℃ and decant the liquid agarose gel into an agarose gel tank. Add 8 μL EB into the liquid agarose gel and place the electrophoresis comb.
2. Load the gel: Place the solid agarose gel in an electrophoresis device. Add 10xDNA Loading Buffer in the DNA sample, mix them up gently, and carefully pipette the sample into the sample loading chambers.
3. Electrophoresis: Cover the lid of the electrophoresis device, set the electrophoresis time, and start electrophoresis.

## Plasmid transformation

Plasmid transformation is performed to transfer plasmids into the host bacteria. We transform our plasmids into DH5α *E. coli* to amplify them and produce metabolites, into BL21 to testify protein expression of our plasmids.

1. Thaw all reagents on ice. And add 20 μL competent *E. coli* cells with desired DNA.
2. Heat shock at 42℃ for 45s and then cool the mixture on ice for 2min.
3. Add 900 μL liquid SOC medium (without antibiotic) into the mixture and shake culture at 37℃ for 1h.
4. Evenly spread 200 μL liquid culture on a solid culture medium and incubate at 37℃ overnight for colonies forming on the plate.

## Plasmid Miniprep

To extract and purify plasmids, we carried out this based on the Vazyme Mini-prep Kit manual. We have publish [it on protocols.io](https://www.protocols.io/view/plasmid-miniprep-for-2022-dm6gpjwdpgzp/v1).

1. Pellet bacteria culture in a 1.6 mL centrifugation tube at 13,000 rpm for 1 minute. Decant supernatant. The pellet in the tube can be stored in -20 freezer if time not enough for the following steps.
2. Add 250 μL of resuspension solution (P1 with RNase) and vortex. After adding RNase into the P1, P1 is stored in 4 degree refrigerator.
3. Add 250 μL of lysis solution (P2) and invert gently 4-6 times. P2 contains NaOH, and must keep tightly closed.
4. Add 350 μL of neutralization solution (P3) and invert gently 4-6 times. Do not vortex, which will break bacteria genomic DNA and genomic DNA fragments will contaminate purified plasmids.
5. Spin at 13,000 rpm for 10 minutes, at 4 dgree.
6. Take the clear supernatant, without any white protein cloud. Bind the DNA to the column by decanting.
7. Centrifuge at 13,300 rpm for 1 minute.
8. Wash the column with 600 μL of wash solution (PW2) and centrifuge for 1 minute. Remind adding pure ethanol into PW2 as indicated on the bottle.
9. Discard the flow-through, wash again with 600 μL of PW2, and centrifuge for 1 minute. Discard the flow-through and centrifuge the column again for 2 minutes.
10. Keep the cap open and wait until no ethanol smell. Move the column into a new centrifugation tube. Elute the purified DNA with 50 μL of elution buffer (TE; 10 mM Tris-Cl, 1 mM EDTA). Then, centrifuge for 1 minutes. Collect the flow-through, which is the plasmid DNA. Because pET28 based is low-copy plasmid, pre-warm TE to 50 degree helps the elution.

## Preparation of bacterial cell lysate for SDS-PAGE

We have publish [it on protocols.io](https://www.protocols.io/view/preparation-of-bacterial-cell-lysate-for-sds-page-14egn2zwyg5d/v1).

1. Grow the cells overnight, to an OD600 above 1. If IPTG induction needed, at it around OD600 0.2-0.3 (keep IPTG stock solution at -20 freezer).
2. Take one 1 ml of OD600 = 1 from each sample. Correct the sample volume to obtain an equivalent size pellet. For us, as quantified using NanoCym950 nanoparticles (equivalent to the size of *E. coli*), 1 OD600 equals to 10^8 nanoparticles per mL. We estimate each well on SDS-PAGE contains proteins from 2\*10^6 bacteria.
3. Spin down the bacterial cells for 1 min at 13,000 rpm in a microfuge.
4. Aspirate any trace of supernatant with a vacuum line avoiding touching the pellet, but removing all liquid.
5. To each cell pellet, add 250 µL of distilled water, resuspend the pellet. Add 250 µL 2x SDS sample buffer (see below). Heat with a 95 degree heating block for 10 minutes. Store the samples at -20 degree freezer until SDS-PAGE.

Our 2x SDS sample buffer, works for Tris-Cl or MOPS based gels:

* Glycerol 20 ml
* SDS 4 g
* 0.5 M Tris-HCl pH 6.8 stock solution 25 ml
* Bromo Phenol Blue 1 mg; dissolve and volume to 95 ml
* add 5% β-Mercaptoethanol before usage

## SDS-PAGE

SDS-PAGE is performed for the separation of polypeptides and confirms whether our circuits expressed properly.

1. Prepare 10 mL 10% Running Gel solution: Add 4.1 mL ddH2O, 3.3 mL 30% Acrylamide/Bis (29:1 or 37.5:1), 2.5 mL 1.5 M Tris-HCl pH8.8, 100 μL 10% SDS, 50 μL 10% APS, 5 μL TEMED. Mix them thoroughly.
2. Prepare 2ml 4% Stacking Gel solution: Add 1.22 ml ddH2O, 0.26ml 30% Acrylamide/Bis (29:1 or 37.5:1), 0.5 mL 0.5 M Tris-HCl pH6.8, 20 μL 10% SDS, 20 μL 10% APS, 2 μL TEMED. Mix them thoroughly.
3. Prepare the protein sample, as described [above](#preparation-of-bacterial-cell-lysate-for-sds-page).
4. Make the gel: Assemble the gel cassette and make sure it does not leak. Fill the gel cassette with the Running Gel softly and fill up the cassette with ddH2O. Keep it still for 10 - 20 min until the water layer can be observed. Pour out the ddH2O completely and fill up the cassette with Stacking Gel. Insert the comb and take care not to catch bubbles under the teeth. Keep it still.
5. Load the gel: Take off the cassette and assemble the gel running stand. Fill the stand with 1 x SDS running buffer and remove the combs from the gel. Mix up Marker with 1 x SDS Loading. Load 5 μL marker mixture into the wells.
6. Electrophoresis: Cover the lid of the electrophoresis device, and start electrophoresis at 200 V until the dye front is nearly at the bottom of the gel.
7. Stain the gel: Submerge the whole piece of the disassembled gel with 0.1% Coomassie Blue dye for at least 30 min.
8. Destain the gel: Pour out the 0.1% Coomassie Blue dye and wash it using ddH2O. Destain with a destaining solution for 30 min. Change the destaining solution to destain until it is clear.
9. Scan the gel.

## IPTG induction experiment

1. Take 10 mL and 5 mL LB liquid medium in two test tubes, 10 mL one is used as the control group and 5 mL one is for the experiment group. Add 500× antibiotic 20 μL and 10 μL, respectively. Culture 6h. Add 1 mL bacteria solution to the experiment group and divide evenly into two tubes (3 mL each) for IPTG-induced and non-induced groups. Another 1 mL bacteria was used to measure initial OD.
2. Measure OD: Measured OD value once per 30 min and once per 15 min after OD reached 0.2.
3. Induce: When OD value reached about 0.6, add 500 mM IPTG to reach the final concentration of 1 mM. Incubate for 3 hours.

## Preparation of agarose pad for microscopy

Click [here](https://www.protocols.io/view/prepare-agarose-pad-for-microscopy-2022-igem-8epv5jze5l1b/v1) to view our steps to perform fluorescence microscopy for *E. coli*

## Acetone extraction of bacteria pellet for HPLC

Click [here](https://www.protocols.io/view/acetone-extraction-of-bacteria-pellet-for-hplc-202-eq2ly7peelx9/v1) to view how we applied HPLC on bacterial products.

{% endblock %}

{% extends "layout.html" %}
{% block page\_author %}the Team{% endblock %}
{% block title %}Results{% endblock %}
{% block lead %}This is an empty page.{% endblock %}
{% block page\_content %}

Go to [https://2022.igem.wiki/fudan/description](/fudan/description)

Go to [https://2022.igem.wiki/fudan/engineering](/fudan/engineering)

Go to [https://2022.igem.wiki/fudan/proof-of-concept](/fudan/proof-of-concept)

Go to [https://2022.igem.wiki/fudan/parts](/fudan/parts)

Go to [https://2022.igem.wiki/fudan/contribution](/fudan/contribution)

{% endblock %}