Team:Mingdao/Practices
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Biobrick Map- Click link to go to biobrick description
Part Registry Name Attribute Part (E-X-Part-S-P) Part Length Gene Gene Length
pSB1C3-Plac-NB (Part: BBa_K1256001)
BENEFIT: cloning genes with NheI & BamHI sites, easily exchange RFP gene
BIOBRICK LIGATION:
- “X-P” ligated to “S-P” ( X, S are compatible): standard part
- “E-N” ligated to “E-X” (N, X are compatible): to exchange front part (Fig 1. A)
- “N-P” ligated to “S-P” (N, S are compatible): to exchange back part (Fig 1. B)
Fig 1. BioBrick “part in part” (image below)
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pSB1C3-Plac-NB-Cm-MN (BBa_K1256002)
BENEFIT: cloning genes with MfeI & NsiI sites under CmR cassette
BIOBRICK LIGATION:
- “M-Ns” ligated to “E-P” ( M, E are compatible; Ns, P are compatible)
pSB1C3-Plac-SS-NB (Part: BBa_K1256003)
Secretion signal (SS): OmpA SS from E. coli
BENEFIT: cloning genes with NheI & BamHI sites, easily exchange RFP gene
BIOBRICK LIGATION:
- “X-P” ligated to “S-P” or “N-P” ligated to “E-X” (X, S, N are compatible)
pSB1C3-Plac-SS-Cecropin-MprF (BBa_K1256004)
MprF of Bacillus subtilis (DB2)added a RBS (BBa_B0034)was amplified by PCR and cloned into the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) using NheI and BamHI sites. Next, the cds of cecropin was synthesized on the primers and subjeted to Gibson Assembly to make this plasmid. The amino acid sequences of cecropin was obtained from Sigma-Aldrich, and the nucleotide sequence was optimized for engineering Escherichia coli using the codon optimization tool provided by Integrated DNA Technologies (IDT).
pSB1C3-Plac-SS-Magainin-MprF (BBa_K1256005)
MprF of Bacillus subtilis (DB2)added a RBS (BBa_B0034)was amplified by PCR and cloned into the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) using NheI and BamHI sites. Next, the cds of magainin was synthesized on the primers and subjeted to Gibson Assembly to make this plasmid. The amino acid sequences of magainin was obtained from Sigma-Aldrich, and the nucleotide sequence was optimized for engineering Escherichia coli using the codon optimization tool provided by Integrated DNA Technologies (IDT).
Colony PCR was performed to check the plasmid (Right Photo). The cecropin and magainin sequences were further confirmed by sequencing.
pSB1C3-Plac-SS-Protease (BBa_K1256006)
The protease coding sequence (cds) of Stenotrophomonas maltophilia K279a was obtained from KEGG Genes Database. The gene was cut by NheI and BamHI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003). The cds can be easily exchanged with NheI and BamHI. However, PstI site can no longer be used in this plasmid due to three more sites on the gene coding region.
pSB1C3-Plac-SS-Lipase (BBa_K1256007)
The lipase coding sequence (cds) of Stenotrophomonas maltophilia K279a was obtained from KEGG Genes Database. The gene was cut by NheI and NsiI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) with NheI and PstI, leaving a scar of NsiI/PsiI in the end.
pSB1C3-Plac-SS-DNase (BBa_K1256008)
The DNase coding sequence (cds) of Stenotrophomonas maltophilia D457 was obtained from KEGG Genes Database. The gene was cut by NheI and BamHI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003). The cds can be easily exchanged with NheI and BamHI. However, PstI site can no longer be used in this plasmid due to two more sites on the gene coding region.
pSB1C3-Plac-SS-RNase (BBa_K1256009)
The RNase coding sequence (cds) of Stenotrophomonas maltophilia K279a was obtained from KEGG Genes Database. The gene was cut by NheI and BglII and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) cut by NheI and BamHI. PstI site can no longer be used in this plasmid due to one more site on the gene coding region.
pSB1C3-Plac-SS-Chitinase (BBa_K1256010)
The chitinase coding sequence (cds) of Stenotrophomonas maltophilia K279a was obtained from KEGG Genes Database. The gene was cut by NheI and NsiI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) with NheI and PstI, leaving a scar of NsiI/PsiI in the end.
pSB1C3-Plac-SS-Glucanase (BBa_K1256011)
The glucanase coding sequence (cds) of Bacillus subtilis 168 was obtained from KEGG Genes Database. The gene was cut by NheI and BamHI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003). The cds can be easily exchanged with other BioBrick parts with NheI/PstI and SpeI/PstI, or be considered as a standard BioBrick parts (E-X-S-P).
Genes of decomposing enzymes were cloned either from Stenotrophomonas maltophilia or Bacillus subtilis. Figure 1 showed the genes were amplified by PCR.
Figure 1. PCR to amplify the genes of decomposing enzymes
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pSB1C3-Plac-RFP-Cm-LacI (BBa_K1256012)
RBS, LacI coding region and terminators (Part:BBa_Q04121) were amplified by PCR and cut by EcoRI and PstI. The PCR-amplified and enzyme-cut product was introduced to the novel designed plasmid backbone of pSB1C3-Plac-NB-Cm-MN ( Part:BBa_K1256002) with MfeI and NsiI. LacI gene is driven by Chloramphenicol promoter. RFP coding region can be exchanged using NheI and BamHI.
Figure 1 showed the result of colony PCR (A) and the RFG gene expression induced by IPTG (B).
(A)(B)
pSB1C3-Plac-ccdB-Cm-LacI (BBa_K1256013)
ccdB coding region was amplified by PCR from BBa_K145151 and exchanged with RFP on the plasmid of BBa_K1256012.
pSB1C3-Pred-ccdB-light sensor (BBa_K1256014)
Gene expression of ccdB (BBa_K145151), a lethal gene, is regulated by the light. The light-regulated system is composed of light sensor (BBa_K1017101) and light-regulated promoter (BBa_K1017301) designed and constructed by NCTU-Formosa in 2013. Light sensor was first amplified by PCR, cut by EcoRI and PstI, and ligated to pSB1C3-Plac-NB-Cm-MN cut by MfeI and NsiI. Then, light-regulated promoter was amplified by PCR, cut by EcoRI and NheI, and ligated to the resulting vector cut by EcoRI and NheI, which maintains the standard BioBrick assembly rule as well as the flexibility of exchanging RFP cds with NheI and BamHI. The ccdB was amplified by PCR using the forward primer with RBS and assembled to the vector with NheI and BamHI.
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We visited the fire station to get more information about the fire extinguishing principles for our project, and we also learned that how terrible the fire disaster could be. The firefighters taught us that there is no possible to prevent all conflagrations, but we can make great effort to prevent or reduce the damage. During this workshop, we not only benefited from it for fire-related knowledge, but also came up with some ideas for our project. Hopefully, our project could let the public understand the fire and its dangerous behavior as well as make good application for fire security in the future.
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During this summer, a special guest, Prof. David Westenberg visited us from Missouri University of Science & Technology, and he is a co-advisor of Missouri-Rolla iGEM team. We introduced to him about our project and demonstrated our fire-burning experiments. We discussed with Prof. David about how we can further not only improve our project, but also outreach to the public. Afterwards, he told some stories about his school and his iGEM team'92s project, which we were all inspired by their creativity and its application. It was a unique and thought-provoking experience for both teams, and we looked forward to more interactions with other iGEMers.
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This summer vacation, we attended to the iGEM Asia conference at NCTU in Taiwan. At first, we were really nervous because there were only three high school teams, while the others were all collegiate teams. However, we became more confident and prepared as we practiced our presentation repeatedly. We came across many amazing ideas and spent a lot of time discussing with other teams during the conference. The iGEM Asia Conference was not only a chance to make friends, but also an experience to share innovative ideas with people.
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At first, a group of Taichung students planned to bike around the countryside of Taiwan which is originally an entertaining trip. Then, we came up with an idea which is popularizing our project and hope to reinforce kids’ concept of bio-technique. More than that, this can also arouse their interest and creativity in this field.
As we go along, we picked several elementary schools randomly and introduce ourselves and what we do to the kids. This is quite an impressive information to those children who have never heard of genetic modification, as a result, they found this attractive and meaningful in improving human’s life.
Once they were told corn can be retransformed and genetcially modified, they came up with many interesting ideas such as shrinking the watermelon or combining some other fruits. This trip takes people in the countryside to reach the modern thought and technology.
National Chung Hsing University
On June 6th, some of our team members visited Chiao Tung University for an iGEM meetup with NCTU-Formosa, while the other members were taking the concept of iGEM and synthetic biology to the public. In order to promote iGEM and synthetic biology to more people, we chose places where Taichung citizens gather for their leisure time such as the plaza in National Chung Hsing University. We talk to people, elder, young and kids we meet in there, from basketball yard to organic farm. We hope that the idea of synthetic biology and iGEM can reach more to the public.
CMP Block Museum of Arts
After stop in Chung Hsing University, our next is to go to the Park Lane by CMP, where is well-known for its shopping malls and green design trail. In order not to disturb people shopping, we promote synthetic biology along the trail. We introduced our project and talked about what we did on fire retardant bio-materials. And we also introduced synthetic biology and iGEM to them.
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Techology Month was held annually in Mingdao High School. During Biotech Week, we introduced our project and showed the students from grades 7 to 12 with some experiments we have learned in bio lab such as DNA gel electrophoresis and plasmid DNA extraction. Hoping that more people could understand synthetic biology and our project. Many students came to our lab, and we demonstrated them how to use the pipette and some lab awareness and skills. With excitement, students are enthusiastic to the experiments, and we have a great time together.
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In recent years, GMO techniques are widely applied in society and GM foods are common to get in the supermarket. However, most of us are not sure whether GM foods are safe. Therefore, we held a forum to discuss the pros and cons on GM foods in school. We were debating whether the society continues or stops the GM foods. One group of students was the affirmative side, and the other was negative side. Both groups showed great debating ability. It was an impressive and fun experience. This forum may make the public aware of the biotech applied in the society.
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On November 1st, 2014, we visited the Academic Sinica, which is one of the top academic institutes in Taiwan. We look around the display of research achievement from labs that inspires us how biotech could be. And we also attended academic symposium, in which we discussed specific topic and expressed our idea. Meanwhile, we went to scientific research lectures, which intrigues our motivation to learn more biotech approaches in the lab.
By the way, we met Dr. Woan-Yuh Tarn in Institute of Biomedical Sciences. We discussed our project and she is interested in our idea. We acquired the materials for gene cloning from her lab. This visit in Sinica has a significant meaning for us and is also the beginning on our iGEM tour.
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We visited iGEM team NTCU-Formosa and met a member, CHAO-DI CHANG to learn the approach about data mining. We also looked around their laboratory and the campus. We asked him about the tricks of searching the bacteria or the protein we need. He told us some useful web resources such as NCBI, UniProtKB and dbPTM. He introduced these webs to us and taught us how to use them and find the target we may need. Also, he showed us how to write a programming code to sort out the data we download. As for we, we told him the idea of our iGEM project and exchanged different ideas and experiences in the previous iGEM Jamboree.
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