iGEM 2016 — Team ZJU-China

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# Team:ZJU-China/Notebook

* [Home](https://2016.igem.org/Team%3AZJU-China)
* Project
  + [Description](https://2016.igem.org/Team%3AZJU-China/Description)
  + [Design](https://2016.igem.org/Team%3AZJU-China/Design)
  + [Experiments](https://2016.igem.org/Team%3AZJU-China/Experiments)
  + [Proof of concept](https://2016.igem.org/Team%3AZJU-China/Proof)
  + [Result](https://2016.igem.org/Team%3AZJU-China/Results)
  + [Notebook](https://2016.igem.org/Team%3AZJU-China/Notebook)
  + [Demonstrate](https://2016.igem.org/Team%3AZJU-China/Demonstrate)
* Parts
  + [Parts](https://2016.igem.org/Team%3AZJU-China/Parts)
  + [Composite Parts](https://2016.igem.org/Team%3AZJU-China/Composite_Part)
  + [Part collection](https://2016.igem.org/Team%3AZJU-China/Part_Collection)
* Modeling
  + [Overview](https://2016.igem.org/Team%3AZJU-China/Model)
  + [Light Control](https://2016.igem.org/Team%3AZJU-China/Model/first)
  + [Infiltration](https://2016.igem.org/Team%3AZJU-China/Model/second)
  + [Oscillation](https://2016.igem.org/Team%3AZJU-China/Model/third)
* [Hardware](https://2016.igem.org/Team%3AZJU-China/Hardware)
* [Safety](https://2016.igem.org/Team%3AZJU-China/Safety)
* Human Practice
  + [HP summary](https://2016.igem.org/Team%3AZJU-China/Human_Practices)
  + [Silver](https://2016.igem.org/Team%3AZJU-China/HP/Silver)
  + [Gold](https://2016.igem.org/Team%3AZJU-China/HP/Gold)
  + [Engagement](https://2016.igem.org/Team%3AZJU-China/Engagement)
* Team
  + [Team](https://2016.igem.org/Team%3AZJU-China/Team)
  + [Collaborations](https://2016.igem.org/Team%3AZJU-China/Collaborations)
  + [Attributions](https://2016.igem.org/Team%3AZJU-China/Attributions)

[ZJU-China](https://2016.igem.org/Team%3AZJU-China)

* [Home](https://2016.igem.org/Team%3AZJU-China)
* Project
  + [Description](https://2016.igem.org/Team%3AZJU-China/Description)
  + [Design](https://2016.igem.org/Team%3AZJU-China/Design)
  + [Experiments](https://2016.igem.org/Team%3AZJU-China/Experiments)
  + [Proof of concept](https://2016.igem.org/Team%3AZJU-China/Proof)
  + [Result](https://2016.igem.org/Team%3AZJU-China/Results)
  + [Notebook](https://2016.igem.org/Team%3AZJU-China/Notebook)
  + [Demonstrate](https://2016.igem.org/Team%3AZJU-China/Demonstrate)
* Parts
  + [Parts](https://2016.igem.org/Team%3AZJU-China/Parts)
  + [Composite Parts](https://2016.igem.org/Team%3AZJU-China/Composite_Part)
  + [Part collection](https://2016.igem.org/Team%3AZJU-China/Part_Collection)
* Modeling
  + [Overview](https://2016.igem.org/Team%3AZJU-China/Model)
  + [Light Control](https://2016.igem.org/Team%3AZJU-China/Model/first)
  + [Infiltration](https://2016.igem.org/Team%3AZJU-China/Model/second)
  + [Oscillation](https://2016.igem.org/Team%3AZJU-China/Model/third)
* [Hardware](https://2016.igem.org/Team%3AZJU-China/Hardware)
* [Safety](https://2016.igem.org/Team%3AZJU-China/Safety)
* Human Practice
  + [HP summary](https://2016.igem.org/Team%3AZJU-China/Human_Practices)
  + [Silver](https://2016.igem.org/Team%3AZJU-China/HP/Silver)
  + [Gold](https://2016.igem.org/Team%3AZJU-China/HP/Gold)
  + [Engagement](https://2016.igem.org/Team%3AZJU-China/Engagement)
* Team
  + [Team](https://2016.igem.org/Team%3AZJU-China/Team)
  + [Collaborations](https://2016.igem.org/Team%3AZJU-China/Collaborations)
  + [Attributions](https://2016.igem.org/Team%3AZJU-China/Attributions)

**Back to top**

![](https://static.igem.org/mediawiki/2016/d/d2/Backtotop.png)

Notebook

![](https://static.igem.org/mediawiki/2016/3/35/ZJUnotebook.jpg)

June

6.15

        Participant: Zhanyu Wang, Han Wan
        **1.Get all the basic parts from Kit plate**
        supD-tRNA ,T7RNAP &PT7

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Transformation | | | | | | |
| Gene of interest | Vector | Ice | 42°C | Ice | 37°C+LB | Plate |
 BBa\_K228001 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | | BBa\_K228000 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM | | BBa\_K1321338 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM | | |

**2.Done the transformation**

6.30

**Transformation**
        Participant： Shisheng Li

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Transformation | | | | | | |
| Gene of interest | Vector | Ice | 42°C | Ice | 37°C+LB | Plate |
 BBa\_I15008 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | | BBa\_I15009 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM | | |

July

7.1

**Liquid Culture**
        Participant: Shisheng Li

|  |  |  |
| --- | --- | --- |
| Liquid Culture | | |
| Name | Medium | Volume/ml |
 BBa\_I15008(pSB1C3) colony\_1 | LB | 10 || BBa\_I15008(pSB1C3) colony\_2 | LB | 10 |
| BBa\_I15008(pSB1C3) colony\_3 | LB | 10 |
| BBa\_I15009(pSB1C3) colony\_1 | LB | 10 |
| BBa\_I15009(pSB1C3) colony\_2 | LB | 10 |
| BBa\_I15009(pSB1C3) colony\_3 | LB | 10 |

7.2

**miniprep**
        Participant: Shisheng Li

|  |  |  |
| --- | --- | --- |
| Miniprep | | |
| Name | 260/280 | C ng/μl |
 BBa\_I15008(pSB1C3) colony\_1 | 1.90 | 34.6 || BBa\_I15008(pSB1C3) colony\_2 | 1.87 | 33.2 |
| BBa\_I15008(pSB1C3) colony\_3 | 1.88 | 58.3 |
| BBa\_I15009(pSB1C3) colony\_1 | 1.75 | 33.0 |
| BBa\_I15009(pSB1C3) colony\_2 | 1.85 | 45.1 |
| BBa\_I15009(pSB1C3) colony\_3 | 1.93 | 49.0 |

**PCR**
        Participant： Shisheng Li

| PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
| --- | --- | --- | --- | --- | --- |
| taq polymerase | 0.5 | Pre Denature | 94 | 180 | / |
| tempalte | 0.5 | Denature | 95 | 30 | 30 |
| 10X Taq buffer | 5 | Annealing | 55 | 30 | 30 |
| primer-forward(10μM) | 2 | Extension | 72 | 45/45 | 30 |
| primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
| dNTP(2.5mM) | 4 | storage | 4 | / | / |
| ddH2O | 36 | / | / | / | / |

    Result:

![](https://static.igem.org/mediawiki/2016/9/9a/NB_0702.jpeg)

7.5

**Construct of double oscillation plasmid Ptd103aiiA-luxS**
        Participant：Jinshi Ran, Hongya Zhu
        • We got luxS gene from 15M, plate 6, distribution kit 2016.
        • We amplified luxS with PCR and added sequences homo with pTD103aiiA to it.
        • We amplified the backbone pTD103aiiA, and added sequence homo with luxS.
        • Using one-step cloning, we assembled them together.
        • We transformed the new constructed plasmid into DH5alpha and cultured it overnight.
        • We screened the positive colony with colony PCR. After conformed, we inoculated it in 5ml LB and cultured it overnight to harvest the pTD103aiiA-luxS.

7.8

**Transformation**
        Participant：Shisheng Li

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Transformation | | | | | | |
| Gene of interest | Vector | Ice | 42°C | Ice | 37°C+LB | Plate |
 cph8 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | | double terminator | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM | | PompC | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM | | |

7.9

**Liquid Culture**
        Participant: Shisheng Li

|  |  |  |
| --- | --- | --- |
| Liquid Culture | | |
| Name | Medium | Volume/ml |
 Cph8 colony\_1 | LB | 10 || Cph8 colony\_2 | LB | 10 |
| double terminator colony\_1 | LB | 10 |
| double terminator colony\_2 | LB | 10 |
| PompC colony\_1 | LB | 10 |
| PompC colony\_2 | LB | 10 |

7.10

**Miniprep**
        Participant: Shisheng Li

|  |  |  |
| --- | --- | --- |
| Miniprep | | |
| Name | 260/280 | C ng/μl |
 Cph8 colony\_1 | 1.90 | 53.3 || Cph8 colony\_2 | 1.87 | 74.1 |
| double terminator colony\_1 | 1.84 | 44.2 |
| double terminator colony\_2 | 1.61 | 50.8 |
| PompC colony\_1 | 1.87 | 43.8 |
| PompC colony\_2 | 2.12 | 37.6 |

**PCR**
        Participant: Shisheng Li

| PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
| --- | --- | --- | --- | --- | --- |
| taq polymerase | 0.5 | Pre Denature | 94 | 180 | / |
| tempalte | 0.5 | Denature | 95 | 30 | 30 |
| 10X Taq buffer | 5 | Annealing | 55 | 30 | 30 |
| primer-forward(10μM) | 2 | Extension | 72 | 180/45/45 | 30 |
| primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
| dNTP(2.5mM) | 4 | storage | 4 | / | / |
| ddH2O | 36 | / | / | / | / |

        Result:
        The result of gel electrophoresis showed that we managed to get the pcr product of Cph8 and PompC but failed in getting double terminator. After we changed the annealing temperature, we finally made it. When sorting out data, we accidently deleted the picture of the gel.

7.16

**1.Measuring and modeling of promoter pluxR**
        Participant: Jinshi Ran, Hongya Zhu
        For better description of single oscillation system, we measured the response intensity of pluxR to AHL. First, we mutated pTD103luxI-sfGFP with knocking gene luxI out with PCR. We named the new plasmid pTD103sfGFP.
We transformed MG1655 strain of E•coil with pTD103sfGFP, inoculated positive colony in 5ml LB with antibiotic 100μg/ml kanamycin and cultured it over night.
We inoculated over night culture with a 1:1000 dilution in 1ml LB and added AHL according a concentration range:10-6~10-11M when the bacteria solution reached an A600nm 0.1.
Cultured it in 37℃ for 8h. We spun it down and concentrated in PBS (pH=7.2), then measured the fluorescence intensity.
        **2.pick colonies and shake**
        Participant：Zhanyu Wang, Han Wan

|  |  |  |
| --- | --- | --- |
| Liquid Culture | | |
| Name | Medium | Volume/ml |
 BBa\_K28001(pSB1C3) colony\_1 | LB | 10 || BBa\_K28001(pSB1C3) colony\_2 | LB | 10 |
| BBa\_K28001(pSB1C3) colony\_3 | LB | 10 |
| BBa\_K28000(pSB1C3) colony\_1 | LB | 10 |
| BBa\_K28000(pSB1C3) colony\_2 | LB | 10 |
| BBa\_K1321338(pSB1C3) colony\_1 | LB | 10 |
| BBa\_K1321338(pSB1C3) colony\_2 | LB | 10 |
| BBa\_K1321338(pSB1C3) colony\_3 | LB | 10 |

7.26

**Find the T7ptag mutation site and design primers for point mutation**
        Participant：Zhanyu Wang, Han Wan

7.27

**Point mutation pcr**
        Participant：Zhanyu Wang, Han Wan

![](https://static.igem.org/mediawiki/2016/9/93/NB_0727part1.jpeg)

![](https://static.igem.org/mediawiki/2016/d/d8/ZJUNBpart.jpg)

August

8.2

**Confirm other parts need in logic gate**
        Participant：Wang Zhanyu, Wan Han

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Transformation | | | | | | |
| Gene of interest | Vector | Ice | 42°C | Ice | 37°C+LB | Plate |
 BBa\_R0010 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | | BBa\_I13453 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM | | BBa\_895006 | pSB1C3 | 30min | 90s | 5min | 1h/1ml | LBCM | |

8.3

**pick colonies and shake**
        Participant：Participant：Zhanyu Wang, Han Wan

|  |  |  |
| --- | --- | --- |
| Liquid Culture | | |
| Name | Medium | Volume/ml |
 BBa\_R0010(pSB1C3) colony\_1 | LB | 10 || BBa\_R0010(pSB1C3) colony\_2 | LB | 10 |
| BBa\_R0010(pSB1C3) colony\_3 | LB | 10 |
| BBa\_I13453(pSB1C3) colony\_1 | LB | 10 |
| BBa\_I13453(pSB1C3) colony\_2 | LB | 10 |
| BBa\_K895006(pSB1C3) colony\_1 | LB | 10 |
| BBa\_K895006(pSB1C3) colony\_2 | LB | 10 |
| BBa\_K895006(pSB1C3) colony\_3 | LB | 10 |

8.4

        Participant：Zhanyu Wang, Han Wan
        **1. Extract the plasmid**
        **2. PCR**

| PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
| --- | --- | --- | --- | --- | --- |
| taq polymerase | 0.5 | Pre Denature | 94 | 180 | / |
| tempalte | 0.5 | Denature | 95 | 30 | 30 |
| 10X Taq buffer | 5 | Annealing | 55 | 30 | 30 |
| primer-forward(10μM) | 2 | Extension | 72 | 45/45 | 30 |
| primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
| dNTP(2.5mM) | 4 | storage | 4 | / | / |
| ddH2O | 36 | / | / | / | / |

**3.Conduct electrophoresis in 1% agarose gel**
        After sequencing, the other parts are all confirmed.

8.5

**Doing overlap PCR, construct the logic gate plasmid.**
        Participant：Zhanyu Wang, Han Wan

8.7-8.9

**Finish the construction of logic gate’s input plasmid(~3500bp,marked with an arrow)**
        Participant： Zhanyu Wang, Han Wan/br>

![](https://static.igem.org/mediawiki/2016/9/97/NB_0807.jpeg)

8.9-8.11

**Finish the construction of logic gate’s output plasmid(~1100bp,marked with arrows)**
        Participant： Zhanyu Wang, Han Wan

![](https://static.igem.org/mediawiki/2016/9/9a/NB_0809.jpeg)

8.11-8.14

**Using infusion cloning to integrate the input plasmid and output plasmid together. (~4500bp,marked with an arrow)**
        Participant： Zhanyu Wang, Han Wan

![](https://static.igem.org/mediawiki/2016/5/5a/NB_0811.jpeg)

        Thus , we finally get our logic gate plasmid with input and output together.

8.15

**Validate the logic gate plasmid. For details, see our protocol in the experiment part.**
        Participant： Zhanyu Wang, Han Wan

8.18

**Validate the logic gate's response concentration.**
        Participant： Zhanyu Wang, Han Wan
        we add 0.5ml of the fresh bacteria culture and appropriate volume of inducer solution to prepare induction system with the concentration gradient of 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4, 10^-3.
        For details, see our protocol in the experiment part.

8.20

**Co-transformation**
        Participant: Shisheng Li

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Co-Transformation | | | | | | |
| Gene of interest | Vector | Ice | 42°C | Ice | 37°C+LB | Plate |
 Two green-TCS plasmids | / | 30min | 90s | 5min | 1h/1ml | LBCM |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | | Two red-TCS plasmids | / | 30min | 90s | 5min | 1h/1ml | LBCM | | |

8.21

**Liquid Culture**
        Participant: Zhanyu Wang, Han Wan

|  |  |  |
| --- | --- | --- |
| Liquid Culture | | |
| Name | Medium | Volume/ml |
 Green TCS\_1 | LB | 5 || Green TCS\_2 | LB | 5 |
| Green TCS\_3 | LB | 5 |
| Green TCS\_4 | LB | 5 |
| Green TCS\_5 | LB | 5 |
| Green TCS\_6 | LB | 5 |
| Green TCS\_7 | LB | 5 |
| Green TCS\_8 | LB | 5 |
 Red TCS\_1 | LB | 5 || Red TCS\_2 | LB | 5 |
| Red TCS\_3 | LB | 5 |
| Red TCS\_4 | LB | 5 |
| Red TCS\_5 | LB | 5 |
| Red TCS\_6 | LB | 5 |
| Red TCS\_7 | LB | 5 |
| Red TCS\_8 | LB | 5 |

8.22

**Fluorescence intensity measuring**
        Participant: Shisheng Li

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Green TCS | | | | |
| RFU | Control | Dark/th> green light | red light | |
 1 | 9074 | 31813 | 39406 | 32336 || 2 | 9022 | 31512 | 39105 | 32491 |
| 3 | 9063 | 31980 | 38535 | 32000 |
| 4 | 9072 | 32396 | 39816 | 32261 |
| 5 | 9130 | 32203 | 39201 | 33136 |
| 6 | 9063 | 32463 | 39829 | 33519 |
| 7 | 9329 | 32370 | 39991 | 33775 |
| 8 | 9192 | 32724 | 39886 | 33168 |
|

|  |  |  |  |
| --- | --- | --- | --- |
| Red light TCS | | | |
| RFU | Control | Dark/th> red light | |
 1 | 369 | 3716 | 3241 || 2 | 248 | 3719 | 3332 |
| 3 | 384 | 3743 | 3263 |
| 4 | 404 | 3761 | 3449 |
| 5 | 408 | 3663 | 3179 |
| 6 | 409 | 3761 | 3416 |
| 7 | 350 | 3761 | 3416 |
| 8 | 357 | 3584 | 3178 |
|

8.23

**Verifying the single oscillation circuit in microfluidics device**
        Participant：Jinshi Ran, Hongya Zhu
        • We co-transformed the MG1655 strain with pTD103aiiA and pTD103luxI-sfGFP
        • We screened the positive colony and inoculated in 50ml LB with antibiotic 100μg/ml ampicillin (Amp) and 50 mgml21 kanamycin (Kan).
        • We spun it down and concentrated it in 5ml of fresh media with surfactant concentration of0.075% Tween20 when the bacteria solution reach an A600nm of 0.05~0.1.
        • We loaded the sample from the cell port while keeping the media port at sufficiently higher pressure than the waste port below to prevent contamination. We manually applied pressure pulses to line to induce a momentary flow change. The flow was then reversed and allow for cell to receive fresh media with 0.075% Tween20 which prevented cells from adhering to the main channels and waste ports.
        • We took pictures of microfluidics in fluorescence microscope every 5 mins and analyzed the fluorescent density with imageJ.

8.25

**Liquid Culture**
        Participant: Shisheng Li

|  |  |  |
| --- | --- | --- |
| Liquid Culture | | |
| Name | Medium | Volume/ml |
 Green TCS\_1 | LB | 5 || Green TCS\_2 | LB | 5 |
| Green TCS\_3 | LB | 5 |
| Green TCS\_4 | LB | 5 |
| Green TCS\_5 | LB | 5 |
| Green TCS\_6 | LB | 5 |
| Green TCS\_7 | LB | 5 |
| Green TCS\_8 | LB | 5 |
 Red TCS\_1 | LB | 5 || Red TCS\_2 | LB | 5 |
| Red TCS\_3 | LB | 5 |
| Red TCS\_4 | LB | 5 |
| Red TCS\_5 | LB | 5 |
| Red TCS\_6 | LB | 5 |
| Red TCS\_7 | LB | 5 |
| Red TCS\_8 | LB | 5 |

8.26

**Fluorescence intensity measuring**
        Participant： Li Shisheng

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Delay of Red TCS | | | | | | |
| RFU | 0h | 1h | 2h | 3h | 4h | 5h |
 1 | 411 | 431 | 439 | 489 | 739 | 1681 || 2 | 438 | 489 | 403 | 471 | 667 | 1690 |
| 3 | 431 | 503 | 541 | 599 | 820 | 1756 |
| 4 | 384 | 489 | 456 | 419 | 833 | 1681 |
| 5 | 506 | 434 | 434 | 530 | 815 | 1553 |
| 6 | 330 | 364 | 426 | 481 | 684 | 1501 |
| 7 | 361 | 391 | 482 | 552 | 739 | 1482 |
| 8 | 395 | 460 | 426 | 574 | 775 | 1641 |

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Green TCS | | | | | | |
| RFU | 0h | 1h | 2h | 3h | 4h | 5h |
 1 | 411 | 516 | 736 | 917 | 1680 | 4200 || 2 | 438 | 554 | 715 | 907 | 1752 | 4190 |
| 3 | 431 | 534 | 723 | 923 | 1712 | 4210 |
| 4 | 384 | 702 | 805 | 839 | 1692 | 4148 |
| 5 | 506 | 590 | 687 | 878 | 1563 | 4045 |
| 6 | 330 | 577 | 639 | 891 | 1580 | 3968 |
| 7 | 361 | 588 | 617 | 860 | 1559 | 3881 |
| 8 | 395 | 569 | 692 | 992 | 1541 | 3849 |

8.27

**PCR**
        Participant: Shisheng Li

| PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
| --- | --- | --- | --- | --- | --- |
| super fidelity DNA polymerase | 1 | Pre Denature | 95 | 180 | / |
| tempalte | 0.5 | Denature | 95 | 15 | 30 |
| 2X buffer | 25 | Annealing | 55 | 15 | 30 |
| primer-forward(10μM) | 2 | Extension | 72 | 80 | 30 |
| primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
| dNTP(10mM) | 1 | storage | 4 | / | / |
| ddH2O | 18.5 | / | / | / | / |

        Template: broken Ptet-cph8 (BBa\_K1886006)

![](https://static.igem.org/mediawiki/2016/c/c6/NB_0827.jpeg)

8.28

    Validate the logic gate's response time. For details, see our protocol in the experiment part.
        Participant：Zhanyu Wang, Han Wan

8.30

**Verifying the double oscillation circuit**
        Participant：Jinshi Ran ,Hongya Zhu
        • We co-transform the MG1655 strain with pTD103 aiiA-luxS、pTD103 luxI-sfGFP and pSB1C3 plsr-YFP.
        • We screened the positive colony and inoculated it in 50ml LB with antibiotic 100μg/ml ampicillin (Amp) and 50 mg/ml kanamycin (Kan)and Chloromycetin(C). We spun it down and concentrated it in 5ml LB when the bacteria solution reached an A600nm of 0.05~0.1.
        • We inoculated it with a 1:1000 dilution in 100ml fresh media. When the bacteria solution reached A600nm 0.2~0.3, we sampled in every 10 min and measured the fluorescence intensity.

September

9.2

Transformation
        Participant: Shisheng Li

| PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
| --- | --- | --- | --- | --- | --- |
| super fidelity DNA polymerase | 1 | Pre Denature | 95 | 180 | / |
| tempalte | 0.5 | Denature | 95 | 15 | 30 |
| 2X buffer | 25 | Annealing | 55 | 15 | 30 |
| primer-forward(10μM) | 2 | Extension | 72 | 80 | 30 |
| primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
| dNTP(10mM) | 1 | storage | 4 | / | / |
| ddH2O | 18.5 | / | / | / | / |

        Template: promoter-ccas

![](https://static.igem.org/mediawiki/2016/f/f1/NB_0902.jpeg)

9.4

**Transformation**
        Participant: Shisheng Li

| PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
| --- | --- | --- | --- | --- | --- |
| super fidelity DNA polymerase | 1 | Pre Denature | 95 | 180 | / |
| tempalte | 0.5 | Denature | 95 | 15 | 30 |
| 2X buffer | 25 | Annealing | 55 | 15 | 30 |
| primer-forward(10μM) | 2 | Extension | 72 | 80 | 30 |
| primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
| dNTP(10mM) | 1 | storage | 4 | / | / |
| ddH2O | 18.5 | / | / | / | / |

        p-sfGFP-ccaR(BBa\_K1886003)

![](https://static.igem.org/mediawiki/2016/e/e5/NB_0904.jpeg)

9.7

**Transformation**
        Participant： Shisheng Li

| PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
| --- | --- | --- | --- | --- | --- |
| super fidelity DNA polymerase | 1 | Pre Denature | 95 | 180 | / |
| tempalte | 0.5 | Denature | 95 | 15 | 30 |
| 2X buffer | 25 | Annealing | 55 | 15 | 30 |
| primer-forward(10μM) | 2 | Extension | 72 | 80 | 30 |
| primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
| dNTP(10mM) | 1 | storage | 4 | / | / |
| ddH2O | 18.5 | / | / | / | / |

        Template: p-HO1-PCYA(BBa\_K1886005)

![](https://static.igem.org/mediawiki/2016/d/d8/NB_0907.jpeg)

9.9

**Validate the plasmid combining logic gate and red light-induced system: PompC+Plac
(3600bp)**

        Participant: Zhanyu Wang, Han Wan

![](https://static.igem.org/mediawiki/2016/8/8c/NB_0909.png)

9.17

**Validate the plasmid combining logic gate and green light-induced system: PcpcG2+Plac**
(3700bp)
        Participant：Zhanyu Wang, Han Wan

![](https://static.igem.org/mediawiki/2016/f/fa/NB_0917.png)

October

10.10

**PCR**
        Participant: Shisheng Li

| PCR | V/μL | Steps | Temperature/μL | Time/s | Cycle |
| --- | --- | --- | --- | --- | --- |
| super fidelity DNA polymerase | 1 | Pre Denature | 95 | 180 | / |
| tempalte | 0.5 | Denature | 95 | 15 | 30 |
| 2X buffer | 25 | Annealing | 55 | 15 | 30 |
| primer-forward(10μM) | 2 | Extension | 72 | 80 | 30 |
| primer-reverse(10μM) | 2 | Final Extension | 72 | 300 | / |
| dNTP(10mM) | 1 | storage | 4 | / | / |
| ddH2O | 18.5 | / | / | / | / |

        Pompc\_CL\_p-lamda\_sfGFP(BBa\_K1886018)
        Ho1\_pcyA\_Pompc\_CL\_Plamda\_sfGFP(BBa\_K1886019)

![](https://static.igem.org/mediawiki/2016/4/41/NB_1010.jpeg)

Notebook for dry lab

5.19

        Started to design the circuit board for the “cipher machine” used in human practice. (Kang)

5.22

        Received the PCB from the factory, started to welding. (Ma)
        Decided to make a computer game that relate to our project.

5.27

        The PCB has some problems, we were working on with through it.

5.30

        Finished the computer game, it looks great! (Li)

6.7

        Started to design the microfluidic device. (Fu)
The electric cipher machine failed to work, Kang made a new PCB.

6.13

        Sent the blueprint of our microfluidic device to the factory.
Started to build our modeling.

6.22

        New PCB arrived, and it worked well. (Ma)

7.13

        Started to edit our wiki.

7.29

        The model for our light control system was finished, the curve is pretty good but not perfect, and we were trying to update the parameters.

8.4

        We compared the results between the modeling and the experiment. The curve is a little bit flat than we expected.

8.29

        The microfluidic device has arrived. The wet lab used it for their experiment.

9.3

        Started to build oscillation model. (Fu)

9.17

        The wiki style has been settled.

9.28

        We finished the oscillation model, but the result didn’t converge.

10.7

        After we changed some of the parameters, the result of our oscillation model finally became reasonable.

![](https://static.igem.org/mediawiki/2016/3/3c/Result.png)

10.14

        Most of our content are finished, started to fulfill the wiki.