iGEM 2018 — Team DLUT_China

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# Team:DLUT China/Notebook

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* [Home](https://2018.igem.org/Team%3ADLUT_China)
* Project
  + [Description](https://2018.igem.org/Team%3ADLUT_China/Description)
  + [Design](https://2018.igem.org/Team%3ADLUT_China/Design)
  + [Experiments](https://2018.igem.org/Team%3ADLUT_China/Experiments)
  + [Results](https://2018.igem.org/Team%3ADLUT_China/Results)
  + [Model](https://2018.igem.org/Team%3ADLUT_China/Model)
  + [Improve](https://2018.igem.org/Team%3ADLUT_China/Improve)
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* PARTS
  + [Parts Overview](https://2018.igem.org/Team%3ADLUT_China/Parts)
  + [Basic parts](https://2018.igem.org/Team%3ADLUT_China/Basic_Part)
  + [Composite Parts](https://2018.igem.org/Team%3ADLUT_China/Composite_Part)
* AWARDS
  + [Model](https://2018.igem.org/Team%3ADLUT_China/Model)
* LAB
  + [Notebook](https://2018.igem.org/Team%3ADLUT_China/Notebook)
  + [InterLab](https://2018.igem.org/Team%3ADLUT_China/InterLab)
  + [Experiments](https://2018.igem.org/Team%3ADLUT_China/Experiments)
* [SAFETY](https://2018.igem.org/Team%3ADLUT_China/Safety)
* Team
  + [Team Members](https://2018.igem.org/Team%3ADLUT_China/_Team_Members)
  + [Collaborations](https://2018.igem.org/Team%3ADLUT_China/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3ADLUT_China/Attributions)
* HUMAN PRACTICES
  + [Human Practices](https://2018.igem.org/Team%3ADLUT_China/Human_Practices)
  + [Public\_Engagement](https://2018.igem.org/Team%3ADLUT_China/Public_Engagement)

## Notebook

#### You'll find that we do experiments almost every day, so please keep scrolling down (๑￫ܫ￩)

* Experiment
  6 Aug 2018

  1 Determination of signal peptide secretion function:
  The plasmid containing the strain was amplified and cultured (at the same time, the strain was preserved), centrifuged, and the supernatant was collected and observed under a fluorescence microscope.

  2 Uric acid threshold system verification: The strain containing the uric acid concentration sensing system was expanded and cultured, and the supernatant was collected by centrifugation and observed under a fluorescence microscope.

  ⅠAnalysis of experimental results:Fluorescence microscopy cannot directly measure the fluorescence intensity of GFP in solution state, the experiment failed

  ⅡReason for failure: experimental design mistakes
* Experiment
  7 Aug 2018

  1 conversion:
  Plasmids containing different signal peptides were transformed into BL21 competent cells and resistant screened using Amp.

  2 fluorescence observation:
  The plasmid-containing strain was expanded and cultured (at the same time, the strain was preserved), and the supernatant was collected by centrifugation, and Abs were measured at 470 nm and 395 nm, respectively.
* Experiment
  8 Aug 2018

  1 conversion:
  Plasmid containing UOX sequence was transformed into BL21 competent cells, and resistance screening was performed using Amp

  2 fluorescence observation:
  The plasmid-containing strain was expanded and cultured (at the same time, the strain was preserved), and the supernatant was collected by centrifugation, and Abs were measured at 470 nm and 395 nm, respectively.
* Experiment
  9 Aug 2018

  Verify the expression of UOX
  1 sonicating the bacterial solution containing the plasmid
  2 The agitated mixture is subjected to agarose gel electrophoresis
* Experiment
  12 Aug 2018

  UOX activity assay
* Experiment
  14 Aug 2018

  1 Verify the expression of UOX:
  Ultrasonic disruption of the bacterial solution containing the plasmid; agarose gel electrophoresis of the disrupted mixture
  2 conversion:
  A plasmid containing four permeation peptides + UOX and a sequence containing only the UOX gene was transformed into BL21 competent cells, and resistance screening was performed using Amp.

  3 UOX activity assay
* Experiment
  15 Aug 2018

  1 Using a plasmid extraction kit to extract plasmids from strains containing four permeation peptides + UOX and only UOX gene sequences
  2 digestion test: cut the four infiltration peptide + UOX and only the UOX gene sequence from the plasmid
* Experiment
  16 Aug 2018

  Agarose gel electrophoresis experiment: electrophoresis of the cut gene fragment, determination of molecular weight, determination of concentration again
* Experiment
  19 Aug 2018

  1.Extraction of five E.coli BL21 plasmid DNA
  2.Agarose gel electrophoresis of the extracted DNA to verify the plasmid
  ![](https://static.igem.org/mediawiki/2018/7/77/T--DLUT_China--notebook_8-19.png)
* Experiment
  21 Aug 2018

  1.Determination of the activity of extracted urate oxidase
  2.The extracted plasmid was digested and ligated, and GFP was replaced with UOX.
  3.Agarose gel electrophoresis of the ligated plasmid
* Experiment
  22 Aug 2018

  Re-extraction of plasmid, digestion, ligation and agarose gel electrophoresis
* Experiment
  24 Aug 2018

  1.PCR amplification of the target plasmid of b1
  2.Agarose gel electrophoresis and observation of the fragment after PCR
  3.Preparation of experimental materials：
  6 conical flasks (100ml), 400ml LB liquid medium, 8 straight tubes, 8 culture dishes
* Experiment
  30 Aug 2018

  Preparation of E.coli Nissle competent cells and plasmid transformation
* Experiment
  31 Aug 2018

  1.After transformation, EcN was cultured in LB liquid medium supplemented with Amp
  2.Culture preservation
  3.Preparation of experimental materials：
  Glycerin, 200ml solid LB medium, 5 culture dishes, 5 straight tubes
  4.Preparation of solid medium plate
* Experiment
  1 Sep 2018

  Inoculatedd DH5a escherichia coli in LB liquid medium and shake overnight.
* Experiment
  2 Sep 2018

  1 It was found that the bacteria preservation in 09.01 was wrong, and what we used was the bacteria preservation of Nissle escherichia coli .
  2 Inoculatedd DH5a escherichia coli in LB liquid medium and shake overnight.
* Experiment
  3 Sep 2018

  1 The plasmid was extracted and the extracted plasmid was done by electrophoresis of agarose gel.
  2 The electrophoresis was failed.
  3 Make an appointment for the related cooperation matters of affiliated high school of Dalian University of Technology, and the appointment time is 09.05.
* Experiment
  4 Sep 2018

  1 The plasmids were extracted, the extracted plasmids were digested. The plasmids were done by electrophoresis.
  2 It failed again.
  ![](https://static.igem.org/mediawiki/2018/0/0a/T--DLUT_China--N2.png)
* Experiment
  5 Sep 2018

  1 The plasmids were extracted. The extracted plasmids were done by electrophoresis and It failed again.
  2 Talk about the related matters with the staff of affiliated high school of Dalian University of Technology.
* Experiment
  6 Sep 2018

  1 The plasmids were extracted. The extracted plasmids were done by electrophoresis and It failed again.
  2 The sequences containing uric acid oxidase were transfected into the escherichia coli competent cells of BL21 and the plate was coated. (Amp was used to screen the colony).
  3 The bacterial colony PCR was performed and the target fragment was found, which proved that the bacteria on the plate were strains containing the target plasmid.
  4 Inoculated escherichia coli in LB liquid medium and shake overnight.
* Experiment
  7 Sep 2018

  1 It was found that the bacteria preservation in 09.01 was wrong, and what we used was the bacteria preservation of Nissle escherichia coli .
  2 Inoculatedd DH5a escherichia coli in LB liquid medium and shake overnight.
* Experiment
  9 Sep 2018

  IPTG was added to the escherichia coli solution of Nissle to induce the expression of GFP.
  No green fluorescence was found under ultraviolet light.
* Experiment
  13 Sep 2018

  1 Five synthetic plasmids were transferred into BL21 escherichia coli competent cells and then coated with flat plates (Amp was used to screen the colonies).
  2 The bacterial colony PCR was performed, and the target fragment was found, which proved that the bacteria on the plate were strains containing the target plasmid.
  3 Inoculated BL21 escherichia coli in LB liquid medium and shake overnight.
* Experiment
  14 Sep 2018

  1 IPTG was added to the BL21 escherichia coli solution of Nissle to induce the expression of GFP.
  2 No green fluorescence was found under ultraviolet light.
* Experiment
  18 Sep 2018

  1 Inoculated BL21 escherichia coli in LB liquid medium and shake it.
  2 IPTG was added to the escherichia coli solution of BL21 to induce the expression of GFP.
  3 No green fluorescence was found under ultraviolet light.
  4 Inoculated DH5a escherichia coli in LB liquid medium and shake overnight.
* Experiment
  19 Sep 2018

  1 Extracting plasmid;
  2 The extracted plasmid was taken with PCR.
* Experiment
  20 Sep 2018

  The extracted plasmids were electrophoresed and the desired bands appeared.
* Experiment
  21 Sep 2018

  1 When the plasmid after PCR was electrophoresed, the expected bands appeared.
  2 But the negative control also produced the expected bands, which may be caused by contamination of the negative control.
  ![](https://static.igem.org/mediawiki/2018/2/20/T--DLUT_China--N3.png)
  Figure 2 PCR
   Took Gel DNA extraction with PCR strip;
  ![](https://static.igem.org/mediawiki/2018/a/aa/T--DLUT_China--N4.png)
  Figure 3 Glue recycling
  ![](https://static.igem.org/mediawiki/2018/e/ec/T--DLUT_China--N5.png)
  Figure 4 Glue recycling
  The extracted plasmid and PCR products were digested by enzyme.
  electrophoresed them and the expected bands appeared.
  ![](https://static.igem.org/mediawiki/2018/e/ed/T--DLUT_China--N6.png)
  Figure 5 Enzyme digestion
  The plasmid concentration was lower than 10ng after Gel DNA extraction.
* Experiment
  23 Sep 2018

  1 Inoculated DH5a escherichia coli in LB liquid medium and shake overnight.
* Experiment
  24 Sep 2018

  1 Extracting plasmid.
  2 The extracted plasmids were electrophoresed and the expected bands appeared.
  3 Inoculated BL21 escherichia coli in LB liquid medium and shaked bacteria;Add IPTG in BL21 escherichia coli bacteria liquid, centrifuged it and collected precipitation of bacteria.
  4 Save it in - 80 ℃ refrigerator.
* Experiment
  25 Sep 2018

  1 Plasmid of Northeastern University arrived;
  2 Plasmids from Northeastern University were transferred into DH5a escherichia coli competent cells and then coated on a flat plate (Amp was used to screen the colonies).
* Experiment
  27 Sep 2018

  1 The transformed plasmids were amplifie.
  2 Took bacteria preservation.
* Experiment
  29 Sep 2018

  Inoculatedd DH5a escherichia coli in LB liquid medium and shaked it overnight.
* Experiment
  30 Sep 2018

  1 Extracting plasmid;
  2 PCR was performed on the extracted plasmid.
  3 Inoculated the BL21 escherichia coli in LB liquid medium and shake the bacteria overnight.

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# Team:DLUT China/Experiments

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* [Home](https://2018.igem.org/Team%3ADLUT_China)
* Project
  + [Description](https://2018.igem.org/Team%3ADLUT_China/Description)
  + [Design](https://2018.igem.org/Team%3ADLUT_China/Design)
  + [Experiments](https://2018.igem.org/Team%3ADLUT_China/Experiments)
  + [Results](https://2018.igem.org/Team%3ADLUT_China/Results)
  + [Model](https://2018.igem.org/Team%3ADLUT_China/Model)
  + [Improve](https://2018.igem.org/Team%3ADLUT_China/Improve)
  + [Demonstrate](https://2018.igem.org/Team%3ADLUT_China/Demonstrate)
* PARTS
  + [Parts Overview](https://2018.igem.org/Team%3ADLUT_China/Parts)
  + [Basic parts](https://2018.igem.org/Team%3ADLUT_China/Basic_Part)
  + [Composite Parts](https://2018.igem.org/Team%3ADLUT_China/Composite_Part)
* AWARDS
  + [Model](https://2018.igem.org/Team%3ADLUT_China/Model)
* LAB
  + [Notebook](https://2018.igem.org/Team%3ADLUT_China/Notebook)
  + [InterLab](https://2018.igem.org/Team%3ADLUT_China/InterLab)
  + [Experiments](https://2018.igem.org/Team%3ADLUT_China/Experiments)
* [SAFETY](https://2018.igem.org/Team%3ADLUT_China/Safety)
* Team
  + [Team Members](https://2018.igem.org/Team%3ADLUT_China/_Team_Members)
  + [Collaborations](https://2018.igem.org/Team%3ADLUT_China/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3ADLUT_China/Attributions)
* HUMAN PRACTICES
  + [Human Practices](https://2018.igem.org/Team%3ADLUT_China/Human_Practices)
  + [Public\_Engagement](https://2018.igem.org/Team%3ADLUT_China/Public_Engagement)

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* [Header](#top)
* [Details](#food)
  + [Signal peptide](#A1)
  + [HucR response threshold](#A2)
  + [Express, urification and analysis of urate oxidase](#A3)
  + [Engineered plasmid](#A4)
  + [Cell permeation peptide experiment](#A5)
  + [Colony number regulation experiment](#A6)
  + [In vitro lethal experiment](#A7)
![](https://static.igem.org/mediawiki/2018/7/76/T--DLUT_China--experiments1.png)

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# Team:DLUT China/Results

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* [Home](https://2018.igem.org/Team%3ADLUT_China)
* Project
  + [Description](https://2018.igem.org/Team%3ADLUT_China/Description)
  + [Design](https://2018.igem.org/Team%3ADLUT_China/Design)
  + [Experiments](https://2018.igem.org/Team%3ADLUT_China/Experiments)
  + [Results](https://2018.igem.org/Team%3ADLUT_China/Results)
  + [Model](https://2018.igem.org/Team%3ADLUT_China/Model)
  + [Improve](https://2018.igem.org/Team%3ADLUT_China/Improve)
  + [Demonstrate](https://2018.igem.org/Team%3ADLUT_China/Demonstrate)
* PARTS
  + [Parts Overview](https://2018.igem.org/Team%3ADLUT_China/Parts)
  + [Basic parts](https://2018.igem.org/Team%3ADLUT_China/Basic_Part)
  + [Composite Parts](https://2018.igem.org/Team%3ADLUT_China/Composite_Part)
* AWARDS
  + [Model](https://2018.igem.org/Team%3ADLUT_China/Model)
* LAB
  + [Notebook](https://2018.igem.org/Team%3ADLUT_China/Notebook)
  + [InterLab](https://2018.igem.org/Team%3ADLUT_China/InterLab)
  + [Experiments](https://2018.igem.org/Team%3ADLUT_China/Experiments)
* [SAFETY](https://2018.igem.org/Team%3ADLUT_China/Safety)
* Team
  + [Team Members](https://2018.igem.org/Team%3ADLUT_China/_Team_Members)
  + [Collaborations](https://2018.igem.org/Team%3ADLUT_China/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3ADLUT_China/Attributions)
* HUMAN PRACTICES
  + [Human Practices](https://2018.igem.org/Team%3ADLUT_China/Human_Practices)
  + [Public\_Engagement](https://2018.igem.org/Team%3ADLUT_China/Public_Engagement)

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* [Header](#top)
* [Details](#food)
  + [Signal peptide](#A1)
  + [The induction concentration of uric acid](#A2)
  + [Express, urification and analysis of urate oxidase](#A3)
  + [Cell penetrating peptide experiment](#A4)
  + [Colony number regulation experiment](#A5)
  + [Invitro lethal experiment](#A6)
![](https://static.igem.org/mediawiki/2018/0/0b/T--DLUT_China--dna%E7%94%B5%E6%B3%B3.png)