iGEM 2016 — Team JNFLS_China

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# Team:JNFLS China/Notebook

* [HOME](https://2016.igem.org/Team%3AJNFLS_China)
* TEAM
  + [members and attributions](https://2016.igem.org/Team%3AJNFLS_China/Attributions)
  + [collaborations](https://2016.igem.org/Team%3AJNFLS_China/Collaborations)
  + [notebook](https://2016.igem.org/Team%3AJNFLS_China/Notebook)
* PROJECT
  + [background](https://2016.igem.org/Team%3AJNFLS_China/Background)
  + [design](https://2016.igem.org/Team%3AJNFLS_China/Design)
  + [experiments and results](https://2016.igem.org/Team%3AJNFLS_China/Experiments%20and%20Results)
  + [proof of concept](https://2016.igem.org/Team%3AJNFLS_China/Proof)
  + [description](https://2016.igem.org/Team%3AJNFLS_China/Description)
  + [demonstrate](https://2016.igem.org/Team%3AJNFLS_China/Demonstrate)
  + [model](https://2016.igem.org/Team%3AJNFLS_China/Model)
* [PARTS](https://2016.igem.org/Team%3AJNFLS_China/Parts)
* [SAFETY](https://2016.igem.org/Team%3AJNFLS_China/Safety)
* OUTLAB
  + [overall HP](https://2016.igem.org/Team%3AJNFLS_China/HP)
  + [silver](https://2016.igem.org/Team%3AJNFLS_China/HP/Silver)
  + [gold](https://2016.igem.org/Team%3AJNFLS_China/HP/Gold)
  + [integrated practices](https://2016.igem.org/Team%3AJNFLS_China/Integrated_Practices)
* [AWARDS](https://2016.igem.org/Team%3AJNFLS_China/Awards)

2016.igem.org/Team:JNFLS\_China

June
**Week 2 (6/11 - 6/17)**

* Searching for references on pubmed. Reading papers and made a decision of our project: micro RNA, macro application. It is about the application of microRNA to the diagnosis of AD
* We had training for safety, including Lab safety, researcher safety and enviroment safety. And we learned some emergency responses, such as how to use the fire extinguisher and fire blanket, and how to use the emergency shower and eye wash.
* Made a plan for our project. Familiar with some technologies from advisors in the lab.

June
**Week 4 (6/25 - 7/1)**

* Transfer GFP plasmid into competent cells, and shaking culture transferred DH5alpha to make the E.coli proliferation and GFP plasmid replication.
* Extract GFP plasmid, electrophoresis for identification GFP plasmid.
* Sequencing the GFP plasmid.
* Repeat shaking culture DH5alpha and continue to extract GFP plasmid. Repeat electrophoresis for identification plasmid.
*  Waiting for synthesized anti-miRNA sponge and restriction endonucleases ordered.

July
**Week 2 (7/9 - 7/15)**

* Culture the transferred DH5alpha on solid medium for 24h.
* Select single clone on the dishes, continue to shaking culture bacteria over night
* Extraction plasmid and identification with electrophoresis
* Repeat linkage of MRG because of failed result last time.
* Sequencing all plasmids we used: RMG, MRG and RGM, in addition to plasmids CKN and CKP which function as negative and positive control, respectively.

July
**Week 4 (7/23 - 7/29)**

* Detect the fluorescence intensity of GFP for 10 kinds of transferred DH5alpha using microplate reader every 1h, untill to culturing bacteria for 20h.
* Detect OD600 value of bacteria every 1h simultaneously to measure bacteria growth speed.
* Analyze experiment data of fluorescence intensity. The result was not as same as we expected probably because that the plasmid and miRNA can not transferred into bacteria simultaneously.
* We planed to modify our experiment protocols.
* we were invited by Shenzhen\_SFLS team to Guangdong Shenzhen for meet-up. So we started to prepare the poster and PPT both of which are used for the meetup in Shenzhen Foreign Language School

August
**Week 2 (8/6 - 8/12)**

* Shaking culture 5 kinds of transferred bacteria.
* Prepare the competent cells using bacteria containing 5 plasmids: CKN, CKP, MRG, RMG and RGM.
* Transfer the irrelevant miRNA (NC) and miRNA-34c, respectively, into 5 kinds of competent cells above. There are 10 experiment groups altogether.
* Detect the fluorescence intensity of GFP and OD600 value for 10 kinds of transferred DH5alpha using microplate reader every 1h, untill to culturing bacteria for 20h. All processes are similar with last time.

August
**Week 4 (8/20 - 8/26)**

* Explore the condition of Flow Cytometry (FCM) using 5 kinds of bacteria without miRNA-34c.
* Transfer miRNA-34c and NC into competent cells containing plasmids. Also designed 10 experiment groups together.
* Detect fluorescence intensity of GFP for 10 groups with FCM only when shaking culture bacteria for 10h.
* Anaysis FCM experiment data.
* o Colaboration: we communicate with FAFU\_China team of Fujian Agriculture and Forestry University of Science and Technology, they put forward a valuable suggestion for us to use flow cytometry to collect more accurate data through offering a detailed protocol of it
* We went to Tiansi Nursing home to interview some old people, introducing prevention methods and treatment of AD, in addition to our iGEM project which is about miRNA application to AD diagnosis. It is a little bit difficult for them to understand that the fluorescent intensity and the miRNA concentration shows a negative correlation.
* We were thinking to improve our project….

September
**Week 2 (9/10 - 9/16)**

* Sequencing 8 kinds of plasmids for further identification.
* Digest 8 kinds of plasmids using proper restriction enzymes.
* Digest synthetic gene fragment containing anti-miRNA-34c sponge and proper restriction enzyme site.
* Electrophoresis to separate the digested gene fragments.
* Re-extract the digested gene fragments from agarose gel using kit.
* Linkage the 8 kinds of plasmids fragments with anti-miRNA-34c sponge fragment.

September
**Week 4 (9/24 - 9/30)**

* Sequencing 8 plasmids when we got 8 kinds of different recombinant plasmids.
* Transformation plasmids, selection single clone, and shaking culture these bacteria.
* Prepare the competent cells using bacteria containing 8 plasmids, respectively.
* Transfering miRNA-34c and NC into these 8 kinds competent cells, respectively. So we have 18 experiment groups, including negative and positive controls.
* Shaking culture all transferred bacteria.
* Detect the fluorescence intensity of GFP and OD600 value for 18 kinds of transferred bacteria using microplate reader every 1h, untill to culturing for 20h.
* Analysis experiment data achieved with microplate reader.

October
**Week 2 (10/8 - 10/14)**

* Continue to write wiki, and complete the judging form.
* Continue to help OUC\_China team to measure some fluorescence data of their one device.
* Continue to verify and develop some previous parts sent by iGEM headquarter.
* Start to detect the function of new plasmid vector containing anti-miRNA34a sponge.

---

June
**Week 1 (6/4 - 6/10)**

* First meeting, brainstroming and discussing what project would we perform.
* Second meeting, continue to discuss the subject of our project.
* Going to the Qilu hospital, reviewing the neurological doctor about Alzheimer’s disease situation, mainly refer to diagnosis and treatment.

June
**Week 3 (6/18 - 6/24)**

* Preparing and autoclaving culture medium (liquid and solid medium), petri dishes, test tubes, EP tubes and tips, etc
* Culture E.coli DH5alpha on the solid medium with petri dishes for 24h.
* Pick single clone, continue to shaking culture E.coli for 3-5h or till the OD600=0.6.
* Prepare the competent cells using E.coli DH5alpha, store at -80℃
* Design plasmid vectors containing reporter gene (GFP) and anti-miRNA. Three different sites were inserted the anti-miRNA-34c respectively. The three sites are: upstream of RNA polymerase binding site (RBS) which is between the promoter and RBS of GFP gene, downstream of RBS which is between the RBS and ORF(opening reading frame, ORF) of GFP gene. The third site is in the 3’-UTR of GFP gene. And they are named as MRG, RMG and RGM, respectively.

July
**Week 1 (7/2 - 7/8)**

* Digest GFP plasmid using proper restriction endonucleases to get different gene fragments for insertion anti-miRNA at different sites
* Digest synthesized gene fragment containing anti-miRNA34c sponge and some restriction enzyme site
* Electrophoresis to separate gene fragments, and re-extract them using kit from agarose gel
* Linkage the GFP-containing plasmid fragments with anti-miRNA-containing DNA fragments, in order to get 3 different plasmids: MRG, RMG and RGM
* Transfer the recombinant genes to trans-DH5alpha (competent cells).

July
**Week 3 (7/16 - 7/22)**

* ransfer 5 plasmids (CKN, CKP, MRG, RMG and RGM) into competent DH5alpha.
* Replicate 5 plasmids in DH5alpha and extract all plasmid using kit, identification with electrophoresis method.
* Measure fluorescence intensity of GFP for DH5alpha which contain 5 plasmids, respectively.
* Co-transfer plasmids and miRNA-34c into competent DH5alpha. We designed 10 experiment groups: CKN and NC (negative control which is an irrelevant miRNA), CKN and miRNA-34c, CKP and NC, CKP and miRNA-34c, MRG and NC, MRG and miRNA-34c, RMG and NC, RMG and miRNA-34c, RGM and NC, and RGM and miRNA-34c.
* Shaking culture 10 kinds of transferred DH5alpha

August
**Week 1 (7/30 - 8/5)**

* Practice our presentation and talk about our poster
* Some players of our team went to communicate friendly with them at the auditorium of Shenzhen Foreign Language School. We talked about stories and experience
* we listened a report regarding the mechanism, application and advantages of transgenic plant by a PhD who gave us some opinions and suggestions for our project.
* We visited the library, assembly hall, statium and a big beautiful campus which are very exciting us in Shenzhen Foreign Language School. We made friends each other.
* Interviewed with consular officer for visa in American Embassy to Beijing.

August
**Week 3 (8/13 - 8/19)**

* Analyzing experiment data. We found the results are similar with that we expected
* Repeat microplate reader experiment in order to get the reliable and stable data.
* Analying experiment data using mathematical model.
* Repeat microplate reader experiment. But this time we use 2 different concentration of miRNA-34c.
* Analyze the effect of different miRNA concentration on the GFP expression.

August
**Week 5 (8/27 - 9/2)**

* Spending a couple of days to prepare 4 posters for publicity to the Museum.
* We went to Shandong Museum of Science and Technology for publicity the common knowledge of AD, the Sythetic Biology, iGEM competition and our project.
* Did survey about the signals, diagnosis and treatment of AD, in addition to the acceptance for our project.
* Analyzing the result of survey.

September
**Week 1 (9/3 - 9/9)**

* We improved our project 1.0 version. We put a NOT gate system in the circuit: using miRNA to control the expression of repressor which is expected to prevent GFP expression. By this transformation, we predict that the fluorescent intensity will change in direct proportion to miRNA concentration, which is easier to comprehend.
* We designed 2 groups of plasmid vectors. One is repressor tetR goup, including 4 kinds of constructive promoters which are different strength: 0.33t, 0.47t, 0.7t and 1.0t. The other one is repressor LacI group, also including 4 different strength contructive promoters: 0.33L, 0.47L, 0.7L and 1.0L.
* Tranfer 8 plasmids containing different strength promoters into competent cells.
* Shaking culture these bacteria containing 8 kinds of different plasmids, respectively.
* Extract 8 kinds of plasmids, electrophoresis for identification.

September
**Week 3 (9/17 - 9/23)**

* Transfer the linkage products into competent cells, respectively.
* Selection positive clone bacteria to shaking culture, and extraction their plasmids.
* Repeat some steps because some expected recombinat plalmids are failed.

October
**Week 1 (10/1 - 10/7)**

* Start to write wiki and prepare some materials for it.
* Repeat to detect the fluorescence intensity of GFP and OD600 value for 18 kinds of transferred bacteria using microplate reade in order to get reliable and stable data.
* Analyze experiment data.
* Colaboration: we communicate with OUC\_China team, and will help OUC\_China team to measure some fluorescence data of their one device.
* Start to verify and develop some previous parts sent by iGEM headquarter.

October
**Week3 (10/15 - 10/21)**

* Continue to write and finish writing wiki
* Continue to detect the function of new plasmid vector containing anti-miRNA34a sponge, based on the same previous protocols.
* Prepare the poster for the Giant Jamboree.
* Prepare presentation for the Giant Jamboree.
* Send our parts to GenScript company that can help us to transfer parts to iGEM HQ
* Expecting to attend the Giant Jamboree.

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