iGEM 2022 — Team mingdao

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## Dec 3, 2022

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## Sep 21, 2022

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[![](https://hatrabbits.com/wp-content/uploads/2017/01/random.jpg)](https://hatrabbits.com/wp-content/uploads/2017/01/random.jpg)
[![](https://picsum.photos/id/237/200/300)](https://picsum.photos/id/23/200/300)
[![](https://source.unsplash.com/random)](https://source.unsplash.com/random)

## Sep 10, 2022

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[![](https://picsum.photos/id/20/300/200)](https://picsum.photos/id/2/200/300)

## Aug 20, 2022

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## Aug 3, 2022

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[![](https://source.unsplash.com/random)](https://source.unsplash.com/random)
[![](https://hatrabbits.com/wp-content/uploads/2017/01/random.jpg)](https://hatrabbits.com/wp-content/uploads/2017/01/random.jpg)
[![](https://picsum.photos/id/237/200/300)](https://picsum.photos/id/237/200/300)

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
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{% 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 main\_image\_url %}https://static.igem.wiki/teams/4150/wiki/engineering-success/hero.png{% endblock %}
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[Characterization](#characterization)

[Improvement](#improvement)

[Phage Engineering](#reporter)

1. [- Reporter](#reporter)
2. [- Vaccine](#vacc_phage_engineering)

[Reference](#ref)

To engineer bacteriophage vaccine vector, we chose a T7 phage-based system because it’s one of the well-studied model bacteriophages with rich lab materials and available assay methods such as T7 promoter, IPTG induction in E. coli BL21 strain, in vitro transcription and translation (TXTL) studies, phage genome sequences and engineering tools1 as well as safe application in clinical trials2.

![](https://static.igem.wiki/teams/4150/wiki/engineering-success/1-design.png)

Prior to engineer T7 bacteriophage, we designed and constructed a composite biobrick part composed of T7 promoter from an existing part, a novel enhanced RBS with a g10 leader sequence, a GFP reporter gene or chicken ovalbumin antigen gene with a 6xHis tag, and a T7 terminator.

![](https://static.igem.wiki/teams/4150/wiki/engineering-success/engineering-1.png)

# CHARACTERIZATION – T7 promoter in an existing part

## The existing part: [Part:BBa\_K3431048](http://parts.igem.org/Part%3ABBa_K3431048)

![](https://static.igem.wiki/teams/4150/wiki/engineering-success/engineering-2.png)

We retransformed E. coli BL21 with the plasmid of T7-RBS-RFP-Tr/pSB1C3 (BBa\_K3431048). Red colors were clearly seen in LB agar plate and LB liquid culture containing 1mM of IPTG compared to non-IPTG as controls after an overnight culture (Fig. 1).

![](https://static.igem.wiki/teams/4150/wiki/engineering-success/engineering-success-photo-3.png)

**Figure 1 |** IPTG induction of RFP expression driven by T7 promoter in E. coli BL21 carrying T7-RBS-RFP-Tr on LB agar plate (A) or LB broth (B) supplemented with 34 μg/mL of chloramphenicol. IPTG concentration is 1mM.

The transformed E. coli BL21 runs in a kinetic mode for 24 hr in the LB broth with or without IPTG. The RFP levels were read by Synergy H1 Hybrid Multi-Mode Reader - BioTek Instruments (Agilent Technologies, Inc.) at an excitation and emission wavelength of 584 nm and 607 nm, respectively. Regardless of some interfering background values in LB media were detected in the first 3 hr, it’s clear that the T7 promoter activities began to be induced around 2 hr and became significantly strong to a level of 1.5-fold induction compared to the controls without IPTG at 12 hr (Fig. 2). The data were consistent with the results seen by naked eyes (Fig. 1).

![](https://static.igem.wiki/teams/4150/wiki/engineering-success/engineering-4.png)

**Figure 2 |** The plasmid of T7-RBS-RFP-Tr/pSB1C3 was transferred into E. coli BL21. Two clones were picked up for IPTG induction assay for 18 hr. RFP levels were read every one hour at an ex/em = 584/607.

# IMPROVEMENT – an enhanced RBS

## The new part: [Part: BBa\_K4150002](http://parts.igem.org/Part%3ABBa_K4150002)

![](https://static.igem.wiki/teams/4150/wiki/engineering-success/engineering-5.png)

## From the existing part: [Part:BBa\_K3431048](http://parts.igem.org/Part%3ABBa_K3431048)

![](https://static.igem.wiki/teams/4150/wiki/engineering-success/engineering-6.png)

In order to increase the expression of protein of interest, we added a
canonical strong RBS ([Part:BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)) with a
leader sequence containing a stem loop from the T7 bacteriophage gene 10
(g10.RBS, [Part:BBa\_K4150000](http://parts.igem.org/Part%3ABBa_K4150000)).

![](https://static.igem.wiki/teams/4150/wiki/engineering-success/engineering-n7.png)

The g10 leader sequence encodes very highly expressed phage proteins, in which 9-base of Epsilon motif exhibits perfect complementary to the 16S RNA of E. coli. This structure connected with the consensus Shine-Dalgarno sequence (SD) can enhance up to 340-fold heterologous gene expression in E. coli3. The g10 leader sequence is also present in several commercially available pET series vectors.

![](https://static.igem.wiki/teams/4150/wiki/engineering-success/engineering-8.png)

We replaced the RBS with g10.RBS in BioBrick Part:BBa\_K3431048 (T7-RBS-RFP-Tr) to create an improved BioBrick Part:BBa\_K4150002 (T7-g10.RBS-RFP-Tr). These two plasmids were transformed into E. coli BL21. In the presence of IPTG, colonies on the LB agar plate and cultures in the LB broth both showed darker red colors for RFP gene expression driven by T7 promoter with g10 leader sequence compared to the original RBS without g10 leader sequence (Fig.3). However, the E. coli BL21 with T7-g10.RBS-RFP-Tr had a higher background level in the absence of IPTG.

![](https://static.igem.wiki/teams/4150/wiki/engineering-success/engineering-n9.png)

Figure 3 | The plasmids as indicated were transformed into E. coli BL21. The E. coli were grown on LB agar plate supplemented with 20 μg/mL of chloramphenicol and 1mM of IPTG. One of the colonies was cultured in LB broth with 34 μg/mL of chloramphenicol in the absence or in the presence of 1mM of IPTG. Inset panel: The plates were observed under a blue LED light box.

Furthermore, the transformed E. coli with T7-g10.RBS-RFP-Tr compared to T7-RBS-RFP-Tr expressed RFP at faster and increased (up to 2.5-fold) fluorescence intensity levels in a time-dependent manner (Fig. 4). The above data demonstrated the g10 leader sequence in front of RBS can significantly enhance gene expression.

![](https://static.igem.wiki/teams/4150/wiki/engineering-success/engineering-10.png)

Figure 4 | The plasmids as indicated were transformed into E. coli BL21. The E. coli were grown in LB broth supplemented with 34 μg/mL of chloramphenicol and 1mM of IPTG for 18 hr. The RFP expression levels were read at ex/em = 584/607 nm in a kinetic mode by Synergy H1 Hybrid Multi-Mode Reader - BioTek Instruments (Agilent Technologies, Inc.). The values of fluorescence intensity were presented by the data with IPTG induction minus the data without induction as background levels.

![](https://static.igem.wiki/teams/4150/wiki/engineering-success/build1.png)

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## What should this page contain?

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* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

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* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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