update des
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continuous-integration/drone/push Build is failing
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This commit is contained in:
parent
0cd700bb93
commit
07bdbc9ee1
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@ -16,10 +16,11 @@ export const enNavbar = navbar([
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children: [
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"/description/README.md",
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"/project/design.md",
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"/project/experiments.md",
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"/engineering/README.md",
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"/project/results.md",
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"/project/model.md",
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"/project/parts.md",
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"/project/hardware.md",
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"/project/safety.md",
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"/contribution/README.md"
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],
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@ -18,10 +18,11 @@ export const enSidebar = sidebar({
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children: [
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"/description/README.md",
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"/project/design.md",
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"/project/experiments.md",
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"/engineering/README.md",
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"/project/results.md",
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"/project/model.md",
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"/project/parts.md",
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"/project/hardware.md",
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"/project/safety.md",
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"/contribution/README.md"
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],
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@ -15,9 +15,9 @@
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border-radius: 20%;
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}
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/* .vp-card {
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min-width: 100%;
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} */
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.vp-card {
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max-width: 100%;
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}
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.vp-card-title {
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font-family: var(--font-family-heading);
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@ -60,6 +60,7 @@ export default hopeTheme(
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echarts: true,
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figure: true,
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flowchart: true,
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footnote: true,
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gfm: true,
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imgLazyload: true,
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imgSize: true,
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@ -5,7 +5,7 @@ category: Team
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tag:
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- Team
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- Attributions
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next: ../team/collaborations.md
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next: ../team/README.md
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---
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@ -8,24 +8,143 @@ tag:
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next: ../project/design.md
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---
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# Inspiration
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## The Problems
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Cutibacterium acnes, the primary bacterium responsible for acne, not only causes post-inflammatory pigmentation spots and acne scars but also triggers inflammation in most adolescents throughout the prolonged course of the disease.
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痤疮是一种常发于青春期的慢性炎症性皮肤病，在青少年人群中普遍存在，为患者身心健康带来较大影响。根据《临床皮肤科杂志.2019.48(9):583-588.》中数据显示，中国人群截面统计痤疮发病率为81%。3%～7%痤疮患者会遗留瘢痕。而目前有许多患者在得了痤疮后并没有及时到医院进行临床诊断，而临床医师对痤疮治疗的选择存在很大差异，有些治疗方法疗效不确定，缺乏循证医学证据支持，个别方法甚至可能对患者造成损害。
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According to the questionnaire we produced, 90.32% of people around us have had acne. When asked about their attitude towards acne, 25.40% of people thought it was super annoying, 18.95% of people hate it, 23.79% thought it was a little annoying, and these people accounted for 68.14% of the total, more than half of the total.
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Acne is a chronic inflammatory skin disease that often occurs in adolescence and is prevalent in the teenagers, bringing a great impact on the physical and mental health of patients. According to the _Journal of Clinical Dermatology.2019.48(9):583-588_, the prevalence of acne in the Chinese population is 81% in cross-section. 3% to 7% of patients with acne will be left with scarring. At present, there are many patients who do not go to the hospital for clinical diagnosis in a timely manner after having acne, and clinicians' choices of acne treatments vary greatly, with some treatments having uncertain efficacy, lacking evidence-based medical evidence to support them, and individual methods may even be damaging to patients.
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::: echarts People's attitude towards the acne.
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Acne can be caused by a variety of reasons, including excessive secretion of androgens and sebum, abnormal keratinization of the follicular cortical gland ducts, and the proliferation of Propionibacterium acnes in the hair follicles. According to incomplete statistics, many adolescents are under pressure from schoolwork, anxiety, etc., which can also subconsciously affect the endocrine function and lead to acne, and many acne sufferers are also anxious about the change in appearance caused by acne itself. Therefore, it is important to treat acne.
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```json
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{
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"legend": {
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"top": "bottom"
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},
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"series": [
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{
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"name": "People's attitude towards the acne.",
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"type": "pie",
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"radius": [20, 100],
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"center": ["50%", "50%"],
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"roseType": "area",
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"itemStyle": {
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"borderRadius": 8
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},
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"data": [
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{
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"value": 25.4,
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"name": "Super Annoying"
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},
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{
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"value": 23.79,
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"name": "A Little Annoying"
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},
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{
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"value": 18.95,
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"name": "Hate It"
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},
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{
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"value": 16.94,
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"name": "It is Normal"
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},
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{
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"value": 13.31,
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"name": "No feelings about it"
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},
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{
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"value": 1.61,
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"name": "Other"
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}
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]
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}
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]
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}
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```
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We also looked at the problems caused by acne. According to our survey, acne has caused facial anxiety to 70.56% of people around us, 62.50% of people have facial discomfort, and other problems such as social disorders and inferiority complex. Only 18.95% people think it doesn't cause trouble.
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痤疮的产生有种种原因，主要以雄性激素及皮脂分泌过多、毛囊皮质腺导管的角化异常和痤疮丙酸杆菌在毛囊内增生是痤疮发病的主要原因。而据不完全统计，很多青少年因为课业压力过大，产生焦虑情绪等也会潜移默化地影响内分泌功能导致产生痤疮，而痤疮的患者也有很多还会对痤疮本身而导致容貌改变感到焦虑。因此，对于痤疮的治疗迫在眉睫。
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::: echarts
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Placeholder: Human Practice 校园问卷
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```js
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option = {
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title: {
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text: 'The Problems Caused by Acne',
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left: 'center'
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},
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tooltip: {
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trigger: 'item'
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},
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series: [
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{
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name: 'Access From',
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type: 'pie',
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radius: '50%',
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data: [
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{ value: 70.56, name: 'Facial Anxiety' },
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{ value: 62.5, name: 'Facial Discomfort' },
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{ value: 16.73, name: 'Treatment Costs Money' },
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{ value: 11.69, name: 'Difficulty Concentration' },
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{ value: 33.06, name: 'Inferiority Complex' },
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{ value: 14.11, name: 'Inconvenient to Make up' },
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{ value: 2.42, name: 'Other Problems' },
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{ value: 18.95, name: 'No Problems' }
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],
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emphasis: {
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itemStyle: {
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shadowBlur: 10,
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shadowOffsetX: 0,
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shadowColor: 'rgba(0, 0, 0, 0.5)'
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}
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}
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}
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]
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};
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```
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:::
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经过我们对目前市面上痤疮相关护肤品的调查，大部分产品采用物理和化学方法对痤疮进行缓解，而我们采用了生物技术，运用工程菌支撑了更加天然的痤疮缓解产品。我们相信这种新颖、方便的技术将为市场带来新的变化，并带来巨大的社会和经济效益。
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To sum up, we think acne is a common problem in the population, and not just among teenagers. Although the exact pathogenesis of acne has not been fully elucidated, existing data suggests that there are multiple factors contributing to its development, including genetic predisposition, excessive sebum production, abnormal keratinization of hair follicle sebaceous ducts, proliferation of Cutibacterium acnes, inflammation, and immune reactions. Common treatment methods for acne include topical medications, systemic drugs, phototherapy, chemical peels, and microneedle therapy. Topical medications often involve the use of antibiotics (such as erythromycin) or antimicrobial agents (such as tretinoin), which can achieve certain therapeutic effects but may lead to bacterial resistance and side effects like skin itching and redness[^1]. Although laser therapy for acne yields quick results, it is associated with strong adverse reactions such as pain, erythema, and pigmentation changes. Other methods have shown some efficacy but have not adequately addressed issues related to cost-effectiveness, treatment duration, and convenience[^2][^3]. In recent years, the understanding of the role played by Cutibacterium acnes in the pathophysiology of acne has undergone a transformation, and an imbalance in the skin microbiome has also been implicated in its occurrence[^4]. This discovery may provide a new perspective for future acne treatments. Therefore, it is evident that acne development involves multiple factors and mechanisms, and actively seeking safe, effective, and reliable treatment methods remains a hot topic in acne management.
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# Project
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## Our inspiration
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Knowing that so many people suffer from acne, and that we suffer from it ourselves, we decided to find a cure. According to the research, porphyrins secreted by Cutibacterium acnes have been identified as the main factor contributing to acne inflammation. Furthermore, this possesses pathways for both porphyrin and vitamin B12 synthesis with shared precursors. When there is more vitamin B12 in the outside environment, the bacteria themselves will obtain vitamin B12 from the outside world and reduce vitamin B12 synthesis, and instead synthesize more porphyrins. Porphyrins are secreted into the external environment, triggering inflammation that leads to acne[^5].
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通过查找文献，我们了解产生痤疮的主要原因之一是由于痤疮丙酸杆菌在毛囊中大量分泌卟啉引发炎症，所以如果我们能减少卟啉，便可以抑制炎症，从而减少痤疮的产生。根据我们查阅的文献，【卟啉和维生素b12在脸上的常驻菌群的合成通路有共同的中间产物（place），如果我们能让中间产物尽可能合成为维生素b12，这样便可以减少卟啉的产生。注意到一类细菌可以吸收维生素b12，我们决定使用一种普遍存在于人体的细菌——大肠杆菌来作为我们的底盘细菌。通过导入VB12转入蛋白基因使其吸收面部的VB12，从而迫使常驻菌群为了维持（稳态？）而将中间产物尽可能合成缺乏的维生素b12，进而减少卟啉的合成【拮抗】，抑制痤疮。
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同时我们导入了VB12转出蛋白基因，从而使大肠杆菌在吸收完面部的VB12后可以进行回收，并导出以便用于对VB12的再利用
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*当然，我们也向工程菌中导入了自杀系统以保证其不会对环境产生危害。】
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## Our Solution
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We designed the experimental project based on the above principles, and hope to have a certain therapeutic effect on acne.
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For this purpose, BJWZ-China utilized arabinose operon in E.coli to regulate the expression of a vitamin B12 transporter called BtuB, maximizing VB12 absorption from the surroundings.
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Reducing the level of vitamin B12 in the environment can effectively decrease porphyrin production and alleviate inflammation.
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Additionally, lactose operon used to control MazEF and ccdB toxin protein expression, ensuring maintenance of biosafety measures.
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This project focuses on addressing concerns related to acne treatment among teenagers while seeking more stable and long-lasting solutions based on skin microbiome conditions.
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## Our Goal
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We hope to achieve two main desired effects through this project.
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First, we hope that engineered bacteria could be put into facial care and treatment products to absorb excessive vitamin B12 in the face, to achieve the effect of reducing inflammation, thereby reducing the occurrence of acne.
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In addition, due to the high tolerance of the selected chassis strain to VB12, we aim to maximize its utility by enabling it not only to absorb and VB12 in acne surroundings but also to release it through other protein channels when needed by the body, thereby maintaining a relatively stable microenvironment of VB12 at the lesion site. This means that when the engineered bacteria have done their job on the human face, they can be recycled and put to more use, thereby contributing to sustainable development.
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We hope to be able to achieve both goals through experimentation and contribute to others.
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## Reference
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> [^1] 李文锐, 林麟. 外用维 A 酸治疗皮肤病的进展 [J]. 国际皮肤性病学杂志，2017，43(3): 133-136
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>
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> [^2] 夏栩琼, 徐慧, 陆雯丽, 等. 30％超分子水杨酸治疗寻常痤疮的疗效观察[J]．中国皮肤性病学杂志, 2019, 33 (5): 616-619
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>
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> [^3] VILLANI A, CARMELA ANNUNZIATA M, ANTONIETTA LUCIANO M, et al. Skin needling for the treatment of acne scarring: a comprehensive review [J]. J Cosmet Dermatol, 2020, 19 (9): 2174-2181
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>
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> [^4] LI C X, YOU Z X, LIN Y X, et al. Skin microbiome differences relate to the grade of acne vulgaris [J]. J Dermatol, 2019, 46 (9): 787-790
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>
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> [^5] Kang D, Shi B, Erfe MC, Craft N, Li H. Vitamin B12 modulates the transcriptome of the skin microbiota in acne pathogenesis. Sci Transl Med. 2015 Jun 24;7(293):293ra103. doi: 10.1126/scitranslmed.aab2009. PMID: 26109103; PMCID: PMC6049814.
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@ -1,101 +1,251 @@
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---
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title: Engineering
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icon: fa-solid fa-gears
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icon: fa-solid fa-vial
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category: Project
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tag:
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- Project
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- Engineering
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prev: ../project/experiments.md
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prev: ../project/design.md
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next: ../project/safety.md
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---
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# Placeholder
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::: echarts
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```json
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|
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{
|
||||
"tooltip": {
|
||||
"trigger": "axis"
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||||
},
|
||||
"legend": {
|
||||
"data": [
|
||||
"0.2",
|
||||
"0.4",
|
||||
"0.6",
|
||||
"0.8",
|
||||
"1.0"
|
||||
]
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},
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"xAxis": {
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||||
"type": "category",
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||||
"boundaryGap": false,
|
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"name": "Time",
|
||||
"data": [
|
||||
"0h",
|
||||
"1h",
|
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"2h",
|
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"3h"
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]
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},
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"yAxis": {
|
||||
"type": "value",
|
||||
"name": "Numbers of Bacterial Colonies"
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},
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||||
"series": [
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{
|
||||
"name": "0.2",
|
||||
"type": "line",
|
||||
"data": [
|
||||
500,
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||||
172.67,
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||||
500,
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145.67
|
||||
]
|
||||
},
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||||
{
|
||||
"name": "0.4",
|
||||
"type": "line",
|
||||
"data": [
|
||||
500,
|
||||
164.33,
|
||||
117.33,
|
||||
160.33
|
||||
]
|
||||
},
|
||||
{
|
||||
"name": "0.6",
|
||||
"type": "line",
|
||||
"data": [
|
||||
321,
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||||
204.67,
|
||||
160,
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||||
67
|
||||
]
|
||||
},
|
||||
{
|
||||
"name": "0.8",
|
||||
"type": "line",
|
||||
"data": [
|
||||
500,
|
||||
256,
|
||||
109.67,
|
||||
94.67
|
||||
]
|
||||
},
|
||||
{
|
||||
"name": "1.0",
|
||||
"type": "line",
|
||||
"data": [
|
||||
450.67,
|
||||
193.67,
|
||||
163.33,
|
||||
169.33
|
||||
]
|
||||
}
|
||||
]
|
||||
}
|
||||
```
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:::
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1. Introduction
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Acne is a chronic inflammatory skin disease of the follicular sebaceous gland units, which is
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predominantly found in adolescents and has a significant psychological and social impact on them.
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The known causes of acne are excessive sebum production, blockage of follicular sebaceous gland
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ducts, bacterial infections and inflammatory reactions, etc. By searching and reading the literature,
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we have learned that too much vitamin B12 has the probability of triggering acne as well (plus
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literature). Porphyrins are known to trigger inflammation. Vitamin B12 and porphyrins are required for
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the survival of Propionibacterium acnes, and they share the same precursor when synthesized in
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Propionibacterium acnes, which means that there is an antagonistic relationship between them, and
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Propionibacterium acnes can obtain vitamin B12 directly from the outside world when the
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concentration of vitamin B12 is high in the external environment, at which time the synthesis of
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vitamin B12 in the body of the strain will decrease In this case, the synthesis of vitamin B12 in the
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strain will decrease and the synthesis of porphyrin will increase, which will trigger inflammation and
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lead to the occurrence of acne when the excess porphyrin is discharged into the external
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environment. We have designed an engineered bacterium that can absorb vitamin B12 from the face
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and hope that it can be used to alleviate porphyrin-induced inflammatory acne.
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2. Experimental materials
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In this study, we mainly constructed the target plasmid in vitro, amplified the required plasmid in
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Escherichia coli (Escherichia coli DH5α), and carried out the relevant transformation by the heat-shock transformation method; after obtaining the target plasmid, we then transformed it into the
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MG1655 strain, and then screened the target strains by colony PCR, enzyme digestion validation and
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so on. The strains and reagent materials involved in this study are shown below:
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2.1 Strain and plasmid
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We used the basidiomycete strain MG1655, this bacterium is used to synthesize vitamin B12 in the
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laboratory, so it is highly tolerant to vitamin B12, and there is no need to worry about the absorption
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of vitamin overdose which will lead to the bacterial viability being weakened.DH5α was used to
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amplify the target plasmid with high efficiency.
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Table 1: Strains and plasmids used in this study
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Name Functional Description Source
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E.coli DH5α Amplification of target plasmid 2nd Lab®
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MG1655 Basidiomycete 2nd Lab®
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pBS Vector Plasmid AZENTA
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pET-28a(+) Vector Plasmid AZENTA
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pACYC184 Vector Plasmid AZENTA
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pSuicide Provide ccdB gene BNU-China
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2.2 Plasmids
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Two plasmids, pBS and pET-28a(+), were used in this study.
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For the pBS plasmid, we inserted the E. coli btuB gene and regulated its expression with an
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arabinose manipulator. btuB is a transmembrane protein that can transport vitamin B12
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into the cell, and we hoped to artificially regulate E. coli's expression of the protein in order to achieve
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its active and efficient uptake of vitamin B12. For the pET-28a(+) plasmid, we inserted the commonly
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used suicide gene ccdB and regulated its expression with a lactose manipulator as a way to
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artificially manipulate bacterial suicide to ensure no environmental leakage is possible.
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2.3 Experimental reagents and media
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We used LB medium with antibiotics and expression inducers added as required. The detailed list is
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given below:
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I. Experimental reagents
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Table 2: Experimental reagents
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Reagent Name Manufacturer
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Restriction endonuclease Takara
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T4 DNALigase Takara
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In-Fusion HD Cloning kit Takara
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Ex-Taq DNA polymerase Takara
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dNTP Takara
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Reagent Name Manufacturer
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DNA Marker Takara
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PCR Mix
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Plasmid Extraction Kit Magen
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PCR Output Recovery DNA Kit Magen
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GeneJET Gum Recovery Kit Magen
|
||||
Ampicillin, sodium salt Sangon Biotech
|
||||
Kanamycin sulfate Sangon Biotech
|
||||
50×TAE Sangon Biotech
|
||||
Vitamin B12 (≥98%) SAITONG
|
||||
LB Broth BaSebio
|
||||
LB Agar BaSebio
|
||||
Vitamin B12 ELISA Kit JINGMEI
|
||||
II. Culture medium
|
||||
LB medium: weigh 40 g of LB agar or LB broth powder, add 800 mL of distilled water, and stir to
|
||||
dissolve the medium, then volume the medium to 1 L, 121 ℃, high-pressure moist heat sterilization
|
||||
for 20 min.
|
||||
III. Antibiotics
|
||||
Purchased ampicillin storage solution concentration of 50 mg/mL, working concentration of 100
|
||||
μg/mL, stored at -20 ℃; kanamycin sulfate solution concentration of 100 mg/mL, working
|
||||
concentration of 50 μg/mL, stored at -20 ℃.
|
||||
3. Experimental Methods
|
||||
3.1 Cultivation and preservation of Escherichia coli
|
||||
Sterilized 50% glycerol in the ultra-clean table was divided into sterilized 2 mL freezing tubes, 1 mL
|
||||
per tube, the mouth of the tubes was loosened and autoclaved again, and the mouth of the tubes was
|
||||
tightened and refrigerated at 4℃ for spare use. Inoculate the purified target strains to be preserved
|
||||
into 5 mL of LB liquid medium containing corresponding antibiotics, incubate at 200 rpm at 37℃
|
||||
overnight, then centrifuge at 5000 rpm for 5 min, discard the supernatant, and then add 1 mL of fresh
|
||||
antibiotic-free LB medium to resuspend the bacterial body, then centrifuge at 5000 rpm for 5 min,
|
||||
wash the bacterial body and discard the supernatant, then add 1 mL of fresh LB medium, resuspend
|
||||
the bacterial body, then add 1 mL of fresh LB medium to resuspend the bacterial body. After that, add
|
||||
1 mL of fresh LB medium, resuspend the organisms and add all of them into the freezing tube, invert
|
||||
and mix well, label with the name of the strain, resistance, production time, and producer, and freeze
|
||||
and store in the refrigerator at -80℃. Prepare LB solid plates with corresponding resistance, and dry the
|
||||
condensed water droplets on the surface of the plates to prevent the formation of fungal moss after
|
||||
the water droplets have not dried and flowed through the colonies during inoculation. With cauterized
|
||||
inoculation ring, take a small amount of bacterial liquid from the glycerol tube, line activation to the
|
||||
plate, 37 ℃ culture 12~18 hours, until the colony grows, pick a single colony and transfer to the
|
||||
corresponding antibiotic-containing LB medium 200 rpm, 37 ℃ culture overnight.
|
||||
3.2 Transformation of E. coli
|
||||
E. coli receptor cells were purchased from 2nd Lab® in 100 µL tubes and the specific transformation
|
||||
steps were as follows:
|
||||
I. Take out the receptor cells from -80℃ refrigerator, put them on ice and wait until they melt, add
|
||||
an appropriate amount of target DNA fragments into the receptor cells, gently flick the tip of the finger
|
||||
tube to mix well, and incubate on ice for 20~30 min.
|
||||
II. After the ice bath, the mixture was heat shocked at 42℃ for 90 s, then quickly placed on ice and
|
||||
left to stand for 2 min. 900 µL of fresh LB liquid medium preheated at 37℃ was added to the mixture,
|
||||
and it was resuscitated for 1 hour at 37℃ and 160 rpm.
|
||||
III. After recovery, centrifuge at 5000 rpm for 5 min, discard the supernatant, resuspend the
|
||||
organisms with 500 µL of fresh medium, and then spread 100 µL of the bacterial solution onto LB
|
||||
plates containing appropriate antibiotics and incubate at 37℃ overnight.
|
||||
3.3 E.coli colony PCR
|
||||
I. After the transformants coated in step 3.2 are cultured overnight to a colony diameter of 0.5 mm,
|
||||
colony PCR can be performed.
|
||||
II. Prepare PCR tubes, preparation plates, primers, gun tips and PCR Mix, etc., and prepare
|
||||
the appropriate system on the lab bench.
|
||||
III. Dip a small amount of single colony of bacteria with a white tip in the ultra-clean bench, stir gently
|
||||
in the PCR system for 5-10 times, and then put the tip on the prepared plate with the same antibiotic
|
||||
added, mark it accordingly, and cultivate it under the corresponding conditions.
|
||||
IV. Determine the correct transformants according to the agarose gel electrophoresis results of PCR
|
||||
products, and then line purification.
|
||||
V. Inoculate the purified transformants into 5 mL of LB medium with appropriate antibiotics, incubate
|
||||
overnight and extract the plasmids, quantify by agarose gel electrophoresis, and then perform
|
||||
enzyme digestion for verification.
|
||||
VI. The correctly verified transformants were expanded and cultured, and then stored in glycerol
|
||||
tubes.
|
||||
3.4 Enzymatic digestion, ligation and PCR system
|
||||
Enzymatic digestion reaction is used to verify whether the target plasmid is correctly ligated, usually
|
||||
using single or double digestion; the template for digestion needs to be determined according to the
|
||||
size of the target band, usually 10 μL system digestion 200-300 ng.
|
||||
Table 3: Enzyme digestion reaction system
|
||||
Reagent Name Reagent Dosage
|
||||
Enzyme A 2 μL
|
||||
Enzyme B 2 μL
|
||||
10× Enzyme Cutting Buffer 1 μL
|
||||
Template 200 ng
|
||||
RNase-free ddH2O Make up 10 μL
|
||||
The target fragment was ligated with the digested vector, and the product after the In-Fusion ligation
|
||||
the reaction was transformed in E. coli, ligating 10 μL of the system as follows:
|
||||
Table 4: Ligation system of gene fragments and vector plasmids
|
||||
Reagent Name Reagent Dosage
|
||||
Linearized plasmid gel recovery product A µL (200 ng)
|
||||
Gene fragment B μL (100 ng)
|
||||
5×In-Fusion HD 2 μL
|
||||
ddH2O Make up 10 μL
|
||||
The system in the PCR tube was mixed, briefly centrifuged, and put into the PCR instrument at 50°C
|
||||
for 15 min. At the end of the reaction, 5 μL of the product was removed for E. coli transformation.
|
||||
The amount of conventional PCR template was 100 ng, and the system was 20 mL; in the case of colony
|
||||
PCR, the enzyme, Buffer and dNTP in the system were by included in the PCR Mix.
|
||||
Table 5: Common PCR reaction system
|
||||
Reagent Name Reagent Dosage
|
||||
10×Ex-taq buffer 2 μL
|
||||
dNTP 2 μL
|
||||
Primer-F/R 1/1 μL
|
||||
Template 1 μL
|
||||
Enzyme 0.2 μL
|
||||
RNase-free ddH2O Make up 20 μL
|
||||
The temperature and time of the PCR program, depending on the enzyme used and the length of the
|
||||
PCR and the annealing temperature of the primers have:
|
||||
Table 6: Reaction procedures of PCR
|
||||
Reaction
|
||||
Temperature Reaction
|
||||
Time Purpose of reaction
|
||||
95℃ 10 min 1. Template pre-denaturation
|
||||
95℃ 30 s 2. Denaturation
|
||||
55℃ 30 s 3. Primer annealing binding template
|
||||
72℃ 4. Chain extension, time depends on chain length
|
||||
and enzyme.
|
||||
15℃ 5. Cooling reaction system
|
||||
2~4. 28~30 cycles
|
||||
4. Experimental steps
|
||||
4.1 Gene sequence search
|
||||
The btuB gene sequence of E.coli MG1655 was obtained by checking the literature NCBI (Gene
|
||||
symbol: btuB; Gene ID: 948468; accessed at https://www.ncbi.nlm.nih.gov/gene/948468).
|
||||
4.2 Synthesis of sequence and construction of expression
|
||||
plasmid
|
||||
The arabinose manipulator requires three genes for arabinose degradation in E. coli: araB, araA and
|
||||
araD, which forms a gene cluster abbreviated as araBAD; the arabinose promoter, ribosome binding
|
||||
site, araBAD and btuB are fused to form a single fragment, i.e., expression of btuB. In addition, the
|
||||
araC gene needs to be synthesized, which is used to regulate arabinose when it is added. which is
|
||||
used to regulate the transcription of araBAD upon the addition of arabinose.
|
||||
A fragment expression of araC was synthesized, retaining the XbaI cleavage site at the 5' end and
|
||||
the SacI cleavage site at the 3' end, and was inserted into the vector plasmid pBS, resulting in the
|
||||
recombinant plasmid pA; a fragment expression of btuB, retaining the XbaI cleavage site at the 5'
|
||||
end, was inserted into the vector plasmid pBS. Expression of btuB, retaining the XbaI cleavage site at
|
||||
the 5' end and the XhoI cleavage site at the 3' end, was inserted into pA, resulting in the recombinant
|
||||
plasmid pBS-BADpromoter-btuB, which can be used to express btuB in MG1655.
|
||||
The ccdB gene in pSuicide, a gift from BNU-China 2023, was used as a reference sequence, and the
|
||||
temperature-sensing suicide switch was retained, while the EcoR I cleavage site was retained at the
|
||||
5' end and the Hind III cleavage site was retained at the 3' end, and then ligated into the vector
|
||||
pACYC184, which was obtained by inserting pBS-BADpromoter-btuB into pA, which can be used to
|
||||
express btuB in MG1655. pACYC184 to obtain the target plasmid pACYC184-ccdB. Theoretically, in
|
||||
the human environment, the temperature is about 37°C; once the bacteria leaks into the environment,
|
||||
this switch is flipped by the low temperature, leading to its suicide.
|
||||
Using pACYC184-ccdB as a template, the optimized ccdB gene was amplified, no longer retaining its
|
||||
temperature control switch, and replaced with a lactose manipulator to regulate ccdB expression. InFusion primers were designed to retain the EcoR I cleavage site at the 5' end and the Hind III
|
||||
cleavage site at the 3' end of the gene, and ligated into the pET-28a(+) vector linearized by the same
|
||||
cleavage to obtain the target plasmid pET-28a(+)-ccdB.
|
||||
4.3 Obtaining the target strains
|
||||
After E. coli transformation, colony PCR and enzymatic verification, we obtained the target strain
|
||||
MG1655+btuB+ccdB, abbreviated as Mbc, which was successfully transfected into pBSBADpromoter-btuB and pET-28a(+)-ccdB plasmids.
|
||||
4.4 Probing the optimal concentration of arabinose to induce
|
||||
btuB expression
|
||||
A single colony of strain Mbc was inoculated in 5 mL of LB medium with both ampicillin and
|
||||
kanamycin, cultured overnight, and then transferred to a new 20 Ml medium and cultured until
|
||||
OD600=0.6.
|
||||
Seven 50 mL conical flasks containing 20 mL of LB liquid medium prepared from the same batch
|
||||
were taken, and the filtered and sterilized arabinose mother liquor was added to make the final
|
||||
concentrations (mass-to-volume ratio: m/v) 0, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and 0.6%, respectively.
|
||||
Subsequently, we inoculated the initial bacterial solution with OD600=0.6 according to 2% of the
|
||||
medium volume and incubated it at 37°C, 200 rpm for 12 h. The next day, we took 3 mL of the
|
||||
bacterial solution and measured the OD600 to confirm that the addition of arabinose did not inhibit
|
||||
the growth of Mbc.
|
||||
Subsequently, we centrifuged the bacterial solution, collected the bacterial body, and commissioned
|
||||
the company to measure the mRNA expression of btuB gene to get the optimal induction
|
||||
concentration of arabinose.
|
||||
4.5 Explore the optimal time and concentration of IPTGinduced ccdB expression
|
||||
A single colony of strain Mbc was inoculated in 5 mL of LB medium with both ampicillin and
|
||||
kanamycin, cultured overnight, and then transferred to a new 20 mL medium and cultured until
|
||||
OD600=0.6.
|
||||
Eight 50 mL conical flasks containing 20 mL LB liquid medium prepared from the same batch were
|
||||
taken, and filtered and sterilized IPTG was added to make the final concentration of 0, 0.2, 0.4, 0.6,
|
||||
0.8, 1.2, and 1.4 mmol/L, respectively, and inoculated at 2% of the volume of the culture solution,
|
||||
incubated at 37°C, 200 rpm, and 100 μL was taken every 1h and applied to the corresponding
|
||||
resistant The incubation was continued for 8h.
|
||||
On the second day, the number of colonies on the culture medium was observed and photographed
|
||||
to make a comparison graph, to find the optimal induction time and concentration.
|
||||
4.6 Assay of the effect on vitamin B12 absorption
|
||||
4.6.1 Setting of the Vitamin B12 Gradient
|
||||
4.6.2 Detection of the absorption effect of vitamin B12
|
||||
5. Experimental results and analysis
|
||||
4.1 Arabinose does not inhibit bacterial growth
|
||||
The OD curves of the above 7 cultures exploring the optimum concentration of arabinose measured
|
||||
after overnight incubation are as follows:
|
||||
[mg1655OdData.xlsx]
|
||||
x-axis: arabinose concentration
|
||||
y-axis: OD value
|
||||
Within the experimental range, the measured OD values of the bacterial solutions showed a
|
||||
fluctuating upward trend with the increase of arabinose concentration, which indicated that arabinose
|
||||
did not inhibit the growth of MG1655.
|
||||
4.2 Optimal concentration of arabinose induction
|
||||
4.3 Optimal time and concentration of IPTG induction
|
||||
4.4 Effect of engineering bacteria on the absorption of vitamin
|
||||
B12
|
||||
|
|
@ -1,13 +1,12 @@
|
|||
---
|
||||
title: Human Practices
|
||||
icon: fa-solid fa-person-dots-from-line
|
||||
category: Human Practices
|
||||
tag:
|
||||
title Human Practices
|
||||
icon fa-solid fa-person-dots-from-line
|
||||
category Human Practices
|
||||
tag
|
||||
- Human Practices
|
||||
---
|
||||
|
||||
|
||||
|
||||
***[后面还要插图片但我今天懒得整了，明天再说]***
|
||||
### I. Summary of Objectives
|
||||
We are committed to solving the harm caused by acne to human beings, from synthetic biology, using new biological means to treat acne. We first designed interviews and conducted market research to collect the opinions of the public on the treatment of acne, which were summarized to make a preliminary plan for our experimental design and extra-product design. After that, we asked the opinions of relevant experts and collected a lot of valuable information, which was very helpful to our experiment, and we would come to discuss and improve the experimental ideas together. We start from the market, observe the problems in life, practically combine with reality, solve people's urgent needs, and save the human beings who are attacked by acne.
|
||||
|
||||
|
|
@ -16,19 +15,23 @@ We are committed to solving the harm caused by acne to human beings, from synthe
|
|||
#### 1.Market Backtesting
|
||||
We investigate products on the market that have high sales, high heat, and high praise, and evaluate them in multiple dimensions, summarizing positive and negative consumer reviews as well as preferences. We will focus on these issues when designing the product.
|
||||
|
||||
###### We Found Some Problems:
|
||||
Other ways to get rid of acne and other brands of acne patches: common products are not friendly to sensitive skin, more expensive, easy to leave scars, the effect varies greatly from person to person, and the way of use is not convenient.
|
||||
###### We Found Some Problems
|
||||
Other ways to get rid of acne and other brands of acne patches common products are not friendly to sensitive skin, more expensive, easy to leave scars, the effect varies greatly from person to person, and the way of use is not convenient.
|
||||
|
||||
***[这里放 .\photo_group\pg1 但是我不会整]***
|
||||
|
||||
|
||||
|
||||
|
||||
|
||||
|
||||
#### 2.Survey of The Public
|
||||
|
||||
###### Online:
|
||||
###### Online
|
||||
We used test questions to categorize the public into a synthetic-biology-based group and a non-synthetic-biology-based group, and set questions for each of them (effects of acne, causes of acne, how to solve the problem, product design suggestions, etc.). These responses helped us to better tailor our products to address the public's concerns.
|
||||
|
||||
We interviewed 138 females, 111 males, and 19 people of other genders, mostly aged 13-22 years old, most of whom said they and others around them had acne, and we learned that people treat acne in a variety of ways, with cleansers and dietary adjustments being the main ones, but there are also many people who choose to squeeze it off themselves or purchase their own medication. Among the people who use drugs, we learned that people will use a variety of drugs, of which the most used are adapalene gel, metronidazole gel, and salicylic acid. The general opinion of the population is that the medications are generally effective, and we conducted a project design and experiment to address this issue. People are relatively well aware of acne and believe that it causes a lot of distress in their lives, including but not limited to facial discomfort and appearance anxiety. People generally hate acne and want more treatment options. This makes it possible for our program to be able to help more people. More people expressed a willingness to try the live bacteria on the face envisioned in our project, while a small percentage of people held resistance to the idea that the live bacteria on the face must be dangerous, so the future development of our project is still open to question, and there are still many issues that need to be considered!
|
||||
|
||||
##### Offline:
|
||||
##### Offline
|
||||
|
||||
###### Preliminary
|
||||
Writing the interview and discussing how to ask questions without being presumptuous.
|
||||
|
|
@ -36,50 +39,63 @@ Writing the interview and discussing how to ask questions without being presumpt
|
|||
###### Practical
|
||||
Ask about acne concerns, product safety concerns, and product suggestions.
|
||||
|
||||
##### Interviews with Pharmacies:
|
||||
We interviewed traditional Chinese pharmacies to learn about the current main ways of treating acne, heard stories from people who suffer from acne, and found that acne has a large negative impact on people and that people desire successful treatment. The main medicines currently available in pharmacies
|
||||
##### Interviews with Pharmacies
|
||||
We interviewed traditional Chinese pharmacies to learn about the current main ways of treating acne, heard stories from people who suffer from acne, and found that acne has a large negative impact on people and that people desire successful treatment. The main medicines currently available in pharmacies.
|
||||
|
||||
|
||||
|
||||
|
||||
|
||||
##### The From
|
||||
In the form of gel, ointment, masks, acne stickers, etc., people choose different forms of products according to different needs, such as: most of the daytime make-up choose acne stickers, and the evening more choice of ointment, masks, etc..
|
||||
In the form of gel, ointment, masks, acne stickers, etc., people choose different forms of products according to different needs, such as most of the daytime make-up choose acne stickers, and the evening more choice of ointment, masks, etc..
|
||||
About our project, the drugstore salesperson said that if we can guarantee that the product is safe, effective, and cost-effective, people will buy it, and she is willing to support our products, in addition, she said that she thinks that the invisible acne stickers are more suitable for adults, while the cartoon shape is more suitable for children.
|
||||
|
||||
|
||||
|
||||
##### Public Perception of Product Safety
|
||||
Of the five passersby interviewed, three were able to accept live bacteria on their faces, two were unable to accept it, one of them thought it was too dangerous, and the other's attitude changed to one of acceptance after learning that E. coli is a skin-resident bacteria. Therefore, we believe that we cannot ignore the concerns of some people about safety issues, so we will indicate the safety of E. coli in the product design.
|
||||
|
||||
##### Conducting Interviews with Hospitals
|
||||
|
||||
|
||||
|
||||
We interviewed dermatologists at two tertiary care hospitals and asked them about their safety concerns and recommendations regarding our program.
|
||||
|
||||
The hospitals asked us questions about
|
||||
- (1) Where, how much, and in what form vitamin B12 is present on the face.
|
||||
- (2) Whether E. coli can survive within the product.
|
||||
|
||||
The doctoral student from the dermatology department of the hospital also gave us some suggestions for improving the experiment:
|
||||
The doctoral student from the dermatology department of the hospital also gave us some suggestions for improving the experiment
|
||||
|
||||
- (1) find a more reliable method to detect the concentration of vitamin B12.
|
||||
- (2) design a vitamin B12-free medium to cultivate E. coli as a control group.
|
||||
- (3) buy antibodies to do western blot experiment to detect the proteins.
|
||||
- (4) design concentration and time gradient experiments.
|
||||
|
||||
|
||||
|
||||
|
||||
|
||||
#### 3.Feedback on The Project
|
||||
|
||||
##### Design:
|
||||
##### Design
|
||||
|
||||
- (1) we will travel to Beijing Normal University to use enzyme labeling to detect Vb12 concentration more accurately;
|
||||
- (2) the experimental and control groups within the range were designed in Ref.
|
||||
|
||||
##### Product design:
|
||||
##### Product Design
|
||||
|
||||
###### Design
|
||||
We designed a total of two products, acne patch, and mask, they have the same structure, from the inside to the outside: a layer of adhesive patch with holes on the top, which facilitates the absorption of Vb12 by engineering bacteria; a layer of semi-transparent membrane, which prevents the engineering bacteria from escaping and at the same time the Vb12 is transmissible; a layer of LB agar medium for cultivating engineering bacteria; a layer of semi-transparent membrane, which prevents the escape of the engineering bacteria; and a layer of cover with a pattern.
|
||||
We designed a total of two products, acne patch, and mask, they have the same structure, from the inside to the outside a layer of adhesive patch with holes on the top, which facilitates the absorption of Vb12 by engineering bacteria; a layer of semi-transparent membrane, which prevents the engineering bacteria from escaping and at the same time the Vb12 is transmissible; a layer of LB agar medium for cultivating engineering bacteria; a layer of semi-transparent membrane, which prevents the escape of the engineering bacteria; and a layer of cover with a pattern.
|
||||
|
||||
###### How to solve the problem of acne
|
||||
###### How to Solve The Problem of Acne
|
||||
The interlayer of the acne stickers or masks on the affected area contains our engineered bacteria, they can absorb the Vb12 in the affected area, resulting in a reduction in the amount of Vb12 in the environment of the affected area, Propionibacterium acnes porphyrin and Vb12 co-precursors more synthesized Vb12, which porphyrin triggers inflammation, so the increased synthesis of Vb12, porphyrin synthesis is reduced, it will be able to achieve anti-inflammatory to eliminate the purpose of the acne.
|
||||
|
||||
###### Audience (acne sufferers) how to use:
|
||||
###### Audience (Acne Sufferers) How to Use
|
||||
Apply the acne patch or mask to the red and swollen pimples, it is recommended to use the acne patch during the day and the mask at night. The engineered bacteria in the product will die after use, so there is no need to worry about environmental pollution.
|
||||
|
||||
###### How to use in other populations
|
||||
###### How to Use in Other Populations
|
||||
Subsequently, we designed to insert the protein that turns out Vb12 in the engineered bacteria, together with the kit that recovers Vb12, and then we can realize the recovery and reuse of Vb12.
|
||||
|
||||
##### Real-life implementation
|
||||
We have designed a series of products with software designed to detect your skin condition, which can recommend the right products for you, and you can buy them on demand. We will be launching other environmentally friendly products like the Vb12 Recycling Box.
|
||||
##### Real-life Implementation
|
||||
We have designed a series of products with software designed to detect your skin condition, which can recommend the right products for you, and you can buy them on demand. We will be launching other environmentally friendly products like the Vb12 Recycling Box.
|
||||
|
|
|
|||
|
|
@ -1,44 +0,0 @@
|
|||
---
|
||||
title: Experiments
|
||||
icon: fa-solid fa-vial
|
||||
category: Project
|
||||
tag:
|
||||
- Project
|
||||
- Experiments
|
||||
---
|
||||
|
||||
|
||||
# 培养基制备
|
||||
|
||||
对于2种培养基，我们根据所用菌的抗性分成三种类型，分别加入四环素粉末，100 mg/mL 的卡纳母液，和 50 mg/mL 的氨苄母液。
|
||||
|
||||
## LB Agar
|
||||
|
||||
|Component|Quality|
|
||||
|:-:|:-:|
|
||||
|蛋白胨（tryptone）|10.00 g/L|
|
||||
|酵母精粉（yeast extract）|5.00 g/L|
|
||||
|食盐（NaCl）|10.00 g/L|
|
||||
|琼脂（agar）|15.00 g/L|
|
||||
|
||||
## LB
|
||||
|
||||
|Component|Quality|
|
||||
|:-:|:-:|
|
||||
|蛋白胨（tryptone）|10.00 g/L|
|
||||
|酵母精粉（yeast extract）|5.00 g/L|
|
||||
|食盐（NaCl）|10.00 g/L|
|
||||
|
||||
四环自杀 氨苄导入 卡纳导出
|
||||
|
||||
## 菌种接种
|
||||
|
||||
对于固体培养基，使用接种环用从平板中挑出单个菌落并放入培养基中。对于液体培养基，使用移液器枪进行转移。
|
||||
|
||||
首先，在固体培养基上在上方快速划出三道横线，然后对接种环进行灼烧，然后旋转 90° 在第一步横线尾端进行同样操作两次，第三次旋转后不对接种环进行灼烧后再重复，注意最后横线尾端不与第一步横线相交。
|
||||
|
||||
Placeholder Image of 接种流程
|
||||
|
||||
## 菌株保存
|
||||
|
||||
培养过夜后，将500μL（或800μL）细菌溶液与EP管中的500μL（或800μL）30%甘油（灭菌）混合。管子保存在-80°C的冰箱中
|
||||
|
|
@ -0,0 +1,17 @@
|
|||
---
|
||||
title: Hardware
|
||||
icon: fa-solid fa-gears
|
||||
category: Project
|
||||
tag:
|
||||
- Project
|
||||
- Hardware
|
||||
---
|
||||
|
||||
## Design
|
||||
We have designed two products, acne sticker and acne mask, they have the same structure but part of different feature. The acne patch is more lightweight and compact for daily use, while the mask has a wider range of functions. Their structures from the inside out are as follows: a layer of adhesive patch with pores on the top to stick the acne sticker and mask firmly on the face, the pores on the top can let the skin breathe freely while letting the Vb12 pass through easily, which is convenient for the engineering bacteria in the inner layer to absorb the Vb12; a layer of semi-transparent membrane, which can prevent the engineering bacteria from escaping, and at the same time the Vb12 can pass through and be absorbed by the engineering bacteria in the next layer; a layer of LB agar medium to cultivate the engineering bacteria; a layer of LB agar medium to cultivate the engineering bacteria. LB agar medium, which is the core of our product. Here Vb12 is absorbed by our engineering bacteria; then another layer of semi-transparent membrane, which can prevent engineering bacteria from escaping to the face and ensure the safety of our products; a layer of cover with pattern, our cover design is one kind of cute pattern, which is more beautiful for customers to put on it and easier to be accepted psychologically, and the other one is Chinese style seal engraving, which is beautiful and at the same time promotes the Chinese traditional culture.
|
||||
|
||||
|
||||
|
||||
|
||||
|
||||
|
||||
|
|
@ -0,0 +1,9 @@
|
|||
---
|
||||
title: Model
|
||||
icon: fa-solid fa-pen
|
||||
category: Project
|
||||
tag:
|
||||
- Project
|
||||
- Model
|
||||
---
|
||||
|
||||
|
|
@ -72,7 +72,7 @@ logo: https://gitlab.igem.org/2022/bjwz-china/-/raw/main/picture/zhanghongjun.jp
|
|||
```
|
||||
```card
|
||||
title: Lei Wang - Primary PI
|
||||
desc: Lei Wang is the Principal and PI of BJWZ-China.
|
||||
desc: Lei Wang is the Principal and PI of BJWZ-China. She possesses a strong inner strength and an abundant passion, providing BJWZ-China with the warmest and the strongest support.
|
||||
logo: https://gitlab.igem.org/2022/bjwz-china/-/raw/252151e6ce9d56d17ec1226de24e84459ae396ac/picture/wanglei.jpg
|
||||
```
|
||||
<!-- color: rgba(197, 225, 254, 0.7)-->
|
||||
|
|
|
|||
Loading…
Reference in New Issue