iGEM 2015 — Team SJTU-BioX-Shanghai

# Team:SJTU-BioX-Shanghai/Notebook

* [home](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai)
* [project](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Description)
  + [description](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Description)
  + [Desalination controller](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Promoters)
  + [Desalination driver](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Transport_Module)
  + [Desalination process](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Design)
  + [Protocol](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Experiments)
* [notebook](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Notebook)
* [safety](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Safety)
* [parts](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Parts)
  + [team parts](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Parts)
  + [Composite parts](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Composite_Part)
* [modeling](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Modeling)
* [human practices](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Practices)
  + [Description](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Practices)
  + [Sea Vs Brackish Water](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Human_Practice/Intake)
  + [sea water restoration](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Human_Practice/Restoration)
  + [Bio & RO](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Human_Practice/Desalination)
  + [Cell water separation](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Human_Practice/Separation)
  + [Public & Government](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Public_Acceptance)
  + [Collaborations](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Collaborations)
* [Achievements](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Achievements)
* [team](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Team)
  + [team members](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Team)
  + [attributions](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai/Attributions)

The Homepage of SJTU-BioX-Shanghai.

Next:  [Home](/Team%3ASJTU-BioX-Shanghai/Home#guide "Team:SJTU-BioX-Shanghai/Home")

# Notebook

## Contents

* [1 Week 1 (July 6 ~)](#Week_1_.28July_6_.7E.29)
  + [1.1 Human Practice](#Human_Practice)
  + [1.2 Construction](#Construction)
    - [1.2.1 July 7](#July_7)
    - [1.2.2 July 9](#July_9)
    - [1.2.3 July 10](#July_10)
    - [1.2.4 July 12](#July_12)
  + [1.3 Transformation](#Transformation)
  + [1.4 Testing](#Testing)
* [2 Week 2 (July 13 ~)](#Week_2_.28July_13_.7E.29)
  + [2.1 Human Practice](#Human_Practice_2)
  + [2.2 Modeling](#Modeling)
  + [2.3 Construction](#Construction_2)
    - [2.3.1 July 13](#July_13)
    - [2.3.2 July 14](#July_14)
    - [2.3.3 July 15](#July_15)
    - [2.3.4 July 16](#July_16)
    - [2.3.5 July 17](#July_17)
    - [2.3.6 July 18](#July_18)
  + [2.4 Transformation](#Transformation_2)
  + [2.5 Testing](#Testing_2)
* [3 Week 3 (July 20 ~)](#Week_3_.28July_20_.7E.29)
  + [3.1 Human Practice](#Human_Practice_3)
  + [3.2 Construction](#Construction_3)
    - [3.2.1 July 21](#July_21)
    - [3.2.2 July 22](#July_22)
    - [3.2.3 July 23](#July_23)
  + [3.3 Transformation](#Transformation_3)
  + [3.4 Testing](#Testing_3)
* [4 Week 4 (July 27 ~)](#Week_4_.28July_27_.7E.29)
  + [4.1 Human Practice](#Human_Practice_4)
  + [4.2 Modeling](#Modeling_2)
  + [4.3 Construction](#Construction_4)
    - [4.3.1 July 29](#July_29)
    - [4.3.2 July 30](#July_30)
    - [4.3.3 July 31](#July_31)
    - [4.3.4 Aug. 1](#Aug._1)
    - [4.3.5 Aug. 2](#Aug._2)
  + [4.4 Transformation](#Transformation_4)
  + [4.5 Testing](#Testing_4)
* [5 Week 5 (August 3 ~)](#Week_5_.28August_3_.7E.29)
  + [5.1 Human Practice](#Human_Practice_5)
  + [5.2 Construction](#Construction_5)
    - [5.2.1 Aug. 3](#Aug._3)
    - [5.2.2 Aug. 5](#Aug._5)
    - [5.2.3 Aug. 6](#Aug._6)
  + [5.3 Transformation](#Transformation_5)
    - [5.3.1 pdark natural transformation](#pdark_natural_transformation)
      * [5.3.1.1 In the noon of Aug. 5](#In_the_noon_of_Aug._5)
      * [5.3.1.2 In the evening of Aug. 7](#In_the_evening_of_Aug._7)
      * [5.3.1.3 Aug. 7](#Aug._7)
    - [5.3.2 pcpcG2 electro-transformation](#pcpcG2_electro-transformation)
      * [5.3.2.1 At the noon of Aug. 8](#At_the_noon_of_Aug._8)
  + [5.4 Testing](#Testing_5)
* [6 Week 6 (August 10 ~)](#Week_6_.28August_10_.7E.29)
  + [6.1 Human Practice](#Human_Practice_6)
  + [6.2 Modeling](#Modeling_3)
  + [6.3 Construction](#Construction_6)
    - [6.3.1 Aug. 10](#Aug._10)
    - [6.3.2 Aug. 11](#Aug._11)
    - [6.3.3 Aug. 12](#Aug._12)
    - [6.3.4 Aug. 13](#Aug._13)
    - [6.3.5 Aug. 14](#Aug._14)
    - [6.3.6 Aug. 16](#Aug._16)
  + [6.4 Transformation](#Transformation_6)
    - [6.4.1 pcpcG2 electro-transformation](#pcpcG2_electro-transformation_2)
      * [6.4.1.1 In the evening of Aug. 10](#In_the_evening_of_Aug._10)
      * [6.4.1.2 In the evening of Aug. 11](#In_the_evening_of_Aug._11)
  + [6.5 Testing](#Testing_6)
* [7 Week 7 (August 17 ~)](#Week_7_.28August_17_.7E.29)
  + [7.1 Human Practice](#Human_Practice_7)
  + [7.2 Construction](#Construction_7)
    - [7.2.1 Aug. 17](#Aug._17)
    - [7.2.2 Aug. 20](#Aug._20)
    - [7.2.3 Aug. 21](#Aug._21)
    - [7.2.4 Aug. 22](#Aug._22)
    - [7.2.5 Aug. 23](#Aug._23)
  + [7.3 Transformation](#Transformation_7)
    - [7.3.1 pdark natural transformation](#pdark_natural_transformation_2)
      * [7.3.1.1 In the evening of Aug 18](#In_the_evening_of_Aug_18)
      * [7.3.1.2 In the evening of Aug. 20](#In_the_evening_of_Aug._20)
    - [7.3.2 pre-experiment of pdark-HR](#pre-experiment_of_pdark-HR)
      * [7.3.2.1 In the evening of Aug. 20](#In_the_evening_of_Aug._20_2)
      * [7.3.2.2 In the evening of Aug. 22](#In_the_evening_of_Aug._22)
      * [7.3.2.3 In the evening of Aug. 23](#In_the_evening_of_Aug._23)
  + [7.4 Testing](#Testing_7)
* [8 Week 8 (August 24 ~)](#Week_8_.28August_24_.7E.29)
  + [8.1 Human Practice](#Human_Practice_8)
  + [8.2 Modeling](#Modeling_4)
  + [8.3 Construction](#Construction_8)
    - [8.3.1 Aug. 25](#Aug._25)
    - [8.3.2 Aug. 26](#Aug._26)
    - [8.3.3 Aug. 27](#Aug._27)
    - [8.3.4 Aug. 28](#Aug._28)
    - [8.3.5 Aug. 29](#Aug._29)
    - [8.3.6 Aug. 30](#Aug._30)
  + [8.4 Transformation](#Transformation_8)
    - [8.4.1 pdark natural transformation](#pdark_natural_transformation_3)
      * [8.4.1.1 In the evening of Aug. 29](#In_the_evening_of_Aug._29)
    - [8.4.2 pcpcG2 electro-transformation](#pcpcG2_electro-transformation_3)
      * [8.4.2.1 In the evening of Aug. 24](#In_the_evening_of_Aug._24)
      * [8.4.2.2 In the evening of Aug. 30](#In_the_evening_of_Aug._30)
    - [8.4.3 pre-experiment of pdark-HR](#pre-experiment_of_pdark-HR_2)
      * [8.4.3.1 Aug. 24](#Aug._24)
      * [8.4.3.2 Aug. 25](#Aug._25_2)
      * [8.4.3.3 Aug. 26](#Aug._26_2)
    - [8.4.4 pdark-GFP electro-transformation](#pdark-GFP_electro-transformation)
      * [8.4.4.1 In the evening of Aug. 24](#In_the_evening_of_Aug._24_2)
      * [8.4.4.2 In the evening of Aug. 26](#In_the_evening_of_Aug._26)
      * [8.4.4.3 In the evening of Aug. 27](#In_the_evening_of_Aug._27)
  + [8.5 Testing](#Testing_8)
* [9 Week 9 (August 31 ~)](#Week_9_.28August_31_.7E.29)
  + [9.1 Human Practice](#Human_Practice_9)
  + [9.2 Modeling](#Modeling_5)
  + [9.3 Construction](#Construction_9)
  + [9.4 Aug. 31](#Aug._31)
  + [9.5 Sep. 2](#Sep._2)
  + [9.6 Sep. 3](#Sep._3)
  + [9.7 Sep. 6](#Sep._6)
  + [9.8 Transformation](#Transformation_9)
    - [9.8.1 pdark natural transformation](#pdark_natural_transformation_4)
      * [9.8.1.1 In the evening of Aug. 31](#In_the_evening_of_Aug._31)
      * [9.8.1.2 In the evening of Sep. 3](#In_the_evening_of_Sep._3)
      * [9.8.1.3 In the evening of Sep. 6](#In_the_evening_of_Sep._6)
    - [9.8.2 pcpcG2 electro-transformation](#pcpcG2_electro-transformation_4)
      * [9.8.2.1 In the evening of Sep. 2](#In_the_evening_of_Sep._2)
      * [9.8.2.2 In the evening of Sep. 4](#In_the_evening_of_Sep._4)
      * [9.8.2.3 In the evening of Sep. 6](#In_the_evening_of_Sep._6_2)
  + [9.9 Testing](#Testing_9)
    - [9.9.1 second desalination test of pdark-HR](#second_desalination_test_of_pdark-HR)
      * [9.9.1.1 Sep. 2](#Sep._2_2)
      * [9.9.1.2 Sep. 4](#Sep._4)
      * [9.9.1.3 Sep. 5](#Sep._5)
      * [9.9.1.4 Sep. 6](#Sep._6_2)
* [10 Week 10 (September 7 ~)](#Week_10_.28September_7_.7E.29)
  + [10.1 Human Practice](#Human_Practice_10)
  + [10.2 Modeling](#Modeling_6)
  + [10.3 Construction](#Construction_10)
  + [10.4 Sep. 7](#Sep._7)
  + [10.5 Sep. 8](#Sep._8)
  + [10.6 Sep. 9](#Sep._9)
  + [10.7 Sep. 10](#Sep._10)
  + [10.8 Sep. 17](#Sep._17)
  + [10.9 Transformation](#Transformation_10)
    - [10.9.1 pdark natural transformation](#pdark_natural_transformation_5)
      * [10.9.1.1 In the evening of Sep. 11](#In_the_evening_of_Sep._11)
    - [10.9.2 pcpcG2 electro-transformation](#pcpcG2_electro-transformation_5)
      * [10.9.2.1 In the evening of Sep. 11](#In_the_evening_of_Sep._11_2)
    - [10.9.3 pdark-GFP electro-transformation](#pdark-GFP_electro-transformation_2)
      * [10.9.3.1 Sep. 8](#Sep._8_2)
  + [10.10 Testing](#Testing_10)
    - [10.10.1 first desalination test of pcpcG2-HR](#first_desalination_test_of_pcpcG2-HR)
      * [10.10.1.1 Sep. 7](#Sep._7_2)
      * [10.10.1.2 Sep. 9](#Sep._9_2)
      * [10.10.1.3 Sep. 10](#Sep._10_2)
      * [10.10.1.4 Sep. 11](#Sep._11)
    - [10.10.2 Detection of Membrane Localization of Halorhodopsin by Immunocytochemistry](#Detection_of_Membrane_Localization_of_Halorhodopsin_by_Immunocytochemistry)
      * [10.10.2.1 Sep. 15](#Sep._15)
      * [10.10.2.2 Sep.15 evening to Sep. 16](#Sep.15_evening_to_Sep._16)

## Week 1 (July 6 ~)

### [![SJTUB hp logo.jpg](/wiki/images/7/79/SJTUB_hp_logo.jpg)](/File%3ASJTUB_hp_logo.jpg) Human Practice

We started main HP project this year. We had already did some communication and discussion with other iGEM team. The initial idea is to tackle our project from different fields, such as law aspect. We start to get in touch with some of our experts.

### [![SJTUB construction logo.jpg](/wiki/images/c/c3/SJTUB_construction_logo.jpg)](/File%3ASJTUB_construction_logo.jpg) Construction

#### July 7

1. Get promotor PcpcB fragment and Kn (kanamycin) fragment respectively from plasmid Bluescript and PRSF by PCR

|  | PcpcB (from pBluescript) | Kn (from pRSF) |
| --- | --- | --- |
| ddwater | 45.5μl | 45.5μl |
| Template | 0.5μl(1ng/50μl) | 0.5μl(1ng/50μl) |
| Primer1(10μM) | 2μl | 2μl |
| Primer2(10μM) | 2μl | 2μl |
| PrimeSTAR Mix | 50μl | 50μl |
| Total volume | 100μl (50μl\*2) | 100μl (50μl\*2) |
|
| ×34 cycle | 98℃ 3min | 98℃ 3min |
| 98℃ 10s | 98℃ 10s |
| 56℃ 5s | 58℃ 5s |
| 72℃ 3s | 72℃ 5s |
| 72℃ 3min | 72℃ 3min |
| 12℃ ∞ | 12℃ ∞ |
| Final concentration(after purification) | 165ng/μl | 225ng/μl |

Gel analyses and purification.

![](https://static.igem.org/mediawiki/2015/a/a0/SJTUB_0708.png)

2. Link aforementioned fragments by overlapping PCR to acquire fragment PcpcB-Kn

|  |  |
| --- | --- |
| ddwater | 90μl |
| Template1(PcpcB) | 1.2μl(50~100ng/50μl) |
| Template2(Kn) | 1.44μl(50~100ng/50μl) |
| Primer1(10μM) | 4μl |
| Primer2(10μM) | 4μl |
| PrimeSTAR mix | 100μl |
| Total volume | 200μl (50μl\*4) |
|
| (round 1)×8 cycle | 98℃ 3min |
| 98℃ 10s |
| 53℃ 5s |
| 72℃ 4s |
| 72℃ 3min |
| 12℃ ∞ |
| (round 2)×35 cycle | 98℃ 3min |
| 98℃ 10s |
| 63℃ 5s |
| 72℃ 7s |
| 72℃ 3min |
| 12℃ ∞ |
| Final concentration(after purification) | Sample1+2: 35ng/μlSample3+4: 54ng/μl |

#### July 9

1. Gel analyses for PcpcB-Kn.
2. DNA purification to get purified fragment PcpcB-Kn.

![](https://static.igem.org/mediawiki/2015/1/19/SJTUB_0709.png)

#### July 10

1. PCR to get fragment PcpcB-Kn-Down. Gel analyses for PcpcB-Kn-Down.
2. Gel extraction to get purified fragment.

![](https://static.igem.org/mediawiki/2015/8/8d/SJTUB_0710.png)

#### July 12

1. Link PcpcB-Kn-Down and Up-GFP (had prepared) by overlapping PCR to get the entire sequence of P\_dark-GFP and gel analyses.
   No correct band is showed.
2. Transformation of E.coli to amplify pBluescript.

### [![SJTUB transformation logo.jpg](/wiki/images/3/31/SJTUB_transformation_logo.jpg)](/File%3ASJTUB_transformation_logo.jpg) Transformation

Learning the method of transformation, including natural transformation and electro-transformation.

### [![SJTUB testing logo.jpg](/wiki/images/6/66/SJTUB_testing_logo.jpg)](/File%3ASJTUB_testing_logo.jpg) Testing

## Week 2 (July 13 ~)

### [![SJTUB hp logo.jpg](/wiki/images/7/79/SJTUB_hp_logo.jpg)](/File%3ASJTUB_hp_logo.jpg) Human Practice

We met Professor Wang and have a long discussion relating to the project feasibility. Because he majors in environment and resource law, we think his questions represent how government and organization will evaluate our project. The plan we had last week can’t cover some of the important problems he raised, so we want to come up with a new plan.

Then, we visited Professor He, an expert working on algae brooms in China. He introduced some of the methods they use to separate cells and also to utilize them. We decided to do some research based on what we learned. Also, we first heard the idea of algae toxin.

### [![SJTUB drylab logo.jpg](/wiki/images/4/41/SJTUB_drylab_logo.jpg)](/File%3ASJTUB_drylab_logo.jpg) Modeling

Discuss the content of modeling

### [![SJTUB construction logo.jpg](/wiki/images/c/c3/SJTUB_construction_logo.jpg)](/File%3ASJTUB_construction_logo.jpg) Construction

#### July 13

1. Overlapping PCR to get the fragment P\_dark-GFP (Annealing temperature changed to a gradience), gel analyses.</br>No correct band is showed.
2. PCR for fragment PcpcB-Kn-Down in the way mentioned before to enlarge backup, gel analyses and purification.</br>Final concentration: 244ng/μl
3. Inoculation of the transformed E.coli.

#### July 14

1. Overlapping PCR for fragment P\_dark-GFP. (Gradient PCR, lengthen annealing and extending time), gel analyses</br>The target band is showed but the concentration is incredibly low and extra bands existed.
2. Extraction of plasmid Bluescript, gel analyses.

#### July 15

1. PCR to get the fragment pCPCG2 from genomic DNA of cyanobacteria and Homoup-R1 from plasmid given. Gel analyses.</br>No positive results are shown. It might be the problem of template quantity or quality.

#### July 16

1. PCR to get the fragment pCPCG2 from genomic DNA of cyanobacteria and Homoup-R1 from plasmid given or the previous fragment UP.

   |  | pCPCG2 | Homoup-R1(from plasmid) | Homoup-R1\*(from DNA fragment) |
   | --- | --- | --- | --- |
   | ddwater | 72μl | 23μl | 22.5μl |
   | Template | 20μl(70ng/50μl) | 0.3μl(1ng/50μl) | 0.5μl(<200ng/50μl) |
   | Primer1 | 4μl | 2μl | 2μl |
   | Primer2 | 4μl | 2μl | 2μl |
   | PrimeSTAR Mix | 100μl | 50μl | 50μl |
   | Total volume | 200μl(50μl\*4) | 100μl(50μl\*2) | 100μl(50μl\*2) |
   |
   | × 34 cycle | 98℃ 3min | 98℃ 3min | 98℃ 3min |
   | 98℃ 10s | 98℃ 10s | 98℃ 10s |
   | (gradient) | 65℃ 5s | 65℃ 5s |
   | 56℃~62℃ 10s | 72℃ 3s | 72℃ 3s |
   | 72℃ 2s | 72℃ 3min | 72℃ 3min |
   | 72℃ 3min | 12℃ ∞ | 12℃ ∞ |
   | 12℃ ∞ |  |  |
   | Final concentration(after purification) | 14.3ng/μl | \ | 29ng/μl |

   \*gradient PCR showed that all annealing temperature can appropriately function.
2. Gel analyses for fragment pCPCG2 and Homoup-R1 and gel extraction.

![](https://static.igem.org/mediawiki/2015/6/6f/SJTUB_0716.png)

#### July 17

1. PCR for fragments Homoup2, Homoup-R1-R2 and HR(including RBS);

   |  | Homoup2 | Homoup-R1-R2 | HR |
   | --- | --- | --- | --- |
   | ddwater | 90μl | 84μl | 92μl |
   | Template | DNA fragment 4μl(<200ng/50μl) | DNA fragment 8μl(116ng/50μl) | Synthetic plasmid 0.1μl(1ng/50μl) |
   | Primer1 | 4μl | 4μl | 4μl |
   | Primer2 | 4μl | 4μl | 4μl |
   | PrimeSTAR Mix | 100μl | 100μl | 100μl |
   | Total volume | 200μl(50μl\*4) | 200μl(50μl\*4) | 200μl(50μl\*4) |
   |
    × 34 cycle | 98℃ 3min | 98℃ 3min | 98℃ 3min || 98℃ 10s | 98℃ 10s | 98℃ 10s |
   | 68℃ 5s | 65℃ 5s | 53℃ 5s |
   | 72℃ 3s | 72℃ 4s | 72℃ 5s |
   | 72℃ 3min | 72℃ 3min | 72℃ 3min |
   | 12℃ ∞ | 12℃ ∞ | 12℃ ∞ |
   | Final concentration (after purification) | 83.5ng/μl | 116.4ng/μl | 114ng/μl |

   Gel analyses and purification/gel extraction.

![](https://static.igem.org/mediawiki/2015/b/b1/SJTUB_0717.png)

1. Overlapping PCR to get the fragments Homoup2-pCPCG2 and P\_dark-HR

   |  | Homoup-pCPCG2 | P\_dark-HR |
   | --- | --- | --- |
   | ddwater | 89μl | 92μl |
   | Template1 | Homoup2 0.3μl(23ng/50μl) | Homoup-R1-R2 0.43μl(50ng/50μl) |
   | Template2 | pCPCG2 3.5μl(50ng/50μl) | HR 0.27μl(31ng/50μl) |
   | Primer1 | 4μl | 4μl |
   | Primer2 | 4μl | 4μl |
   | PrimeSTAR Mix | 100μl | 100μl |
   | Total volume | 200μl(50μl\*4) | 200μl(50μl\*4) |
   |
   | (round 1)×8 cycle | 98℃ 3min | 98℃ 3min |
   | 98℃ 10s | 98℃ 10s |
   | 55℃ 5s | 45℃ 5s |
   | 72℃ 3s | 72℃ 5s |
   | 72℃ 3min | 72℃ 3min |
   | 12℃ ∞ | 12℃ ∞ |
   | 12℃ ∞ | 98℃ 3min | 98℃ 3min |
   | 98℃ 10s | 98℃ 10s |
   | (gradient) | (gradient) |
   | 56℃~68℃ 5s | 59℃~64℃ 5s |
   | 72℃ 3s | 72℃ 8s |
   | 72℃ 3min | 72℃ 3min |
   | 12℃ ∞ | 12℃ ∞ |
   | Final concentration (after purification) | 121.5ng/μl | 187.3ng/μl |

   \*gradient PCR showed that all annealing temperature can appropriately function.</br>Gel analyses and purification/gel extraction.

![](https://static.igem.org/mediawiki/2015/6/64/SJTUB_0717%282%29.png)

#### July 18

1. Overlapping PCR for fragment pCPCG2-HR.

   |  | PcpcG2-HR |
   | --- | --- |
   | ddwater | 89μl |
   | Template1 | Homoup-PcpcG2 1.6μl(50ng/50μl) |
   | Template2 | HR 1.4μl(41ng/50μl) |
   | Primer1 | 4μl |
   | Primer2 | 4μl |
   | PrimeSTAR Mix | 100μl |
   | Total volume | 200μl(50μl\*4) |
   |
   | 200μl(50μl\*4) | 98℃ 3min |
   | 98℃ 10s |
   | 45.5℃ 5s |
   | 72℃ 5s |
   | 72℃ 3min |
   | 12℃ ∞ |
   | 200μl(50μl\*4) | 98℃ 3min |
   | 98℃ 10s |
   | (gradient) |
   | 59℃~63℃ 5s |
   | 72℃ 9s |
   | 72℃ 3min |
   | 12℃ ∞ |
   | Final concentration(after purification) | 145.2ng/μl |

   \*gradient PCR showed that all annealing temperature can appropriately function.</br>Gel analyses and purification/gel extraction.

![](https://static.igem.org/mediawiki/2015/a/a0/SJTUB_0718.png)

1. Overlapping PCR to get the fragments P\_dark-HR-PcpcB and pCPCG2-HR-PcpcB;

   |  | P\_dark-HR-PcpcB | PcpcG2-HR-PcpcB |
   | --- | --- | --- |
   | ddwater | 80μl | 80μl |
   | Template1 | P\_dark-HR 0.4μl(19ng/50μl) | PcpcG2-HR 0.47μl(17ng/50μl) |
   | Template2 | PcpcB 12μl(50ng/50μl) | PcpcB 12μl(50ng/50μl) |
   | Primer1 | 4μl | 4μl |
   | Primer2 | 4μl | 4μl |
   | PrimeSTAR Mix | 100μl | 100μl |
   | Total volume | 200μl(50μl\*4) | 200μl(50μl\*4) |
   |
   | （round 1）×8 cycle | 98℃ 3min | 98℃ 3min |
   | 98℃ 10s | 98℃ 10s |
   | 65℃ 5s | 65℃ 5s |
   | 72℃ 8s | 72℃ 9s |
   | 72℃ 3min | 72℃ 3min |
   | 12℃ ∞ | 12℃ ∞ |
   | (round2) ×33 cycle | 98℃ 3min | 98℃ 3min |
   | 98℃ 10s | 98℃ 10s |
   | 57℃ 5s | 57℃ 5s |
   | 72℃ 12s | 72℃ 13s |
   | 72℃ 3min | 72℃ 3min |
   | 12℃ ∞ | 12℃ ∞ |
   | Final concentration (after purification) | 165.8ng/μl | 170.4ng/μl |

   Gel analyses and purification/gel extraction.

![](https://static.igem.org/mediawiki/2015/9/90/SJTUB_0818%282%29.png)

1. Overlapping PCR to get the whole construction P\_dark-HR\* and pCPCG2-HR\*

   |  | P\_dark-HR\* | PcpcG2-HR\* |
   | --- | --- | --- |
   | ddwater | 88μl | 88μl |
   | Template1 | P\_dark-HR-PcpcB 1.1μl(46ng/50μl) | PcpcG2-HR-PcpcB 1μl(43ng/50μl) |
   | Template2 | PcpcB-Kn 2.9μl(50ng/50μl) | PcpcB-Kn 2.9μl(50ng/50μl) |
   | Primer1 | 4μl | 4μl |
   | Primer2 | 4μl | 4μl |
   | PrimeSTAR Mix | 100μl | 100μl |
   | Total volume | 200μl(25μl\*8) | 200μl(25μl\*8) |
   |
   | （round 1）×8 cycle | 98℃ 3min | 98℃ 3min |
   | 98℃ 10s | 98℃ 10s |
   | 68℃ 8s | 68℃ 8s |
   | 72℃ 12s | 72℃ 12s |
   | 72℃ 3min | 72℃ 3min |
   | 12℃ ∞ | 12℃ ∞ |
   | (round2) ×33 cycle | 98℃ 3min | 98℃ 3min |
   | 98℃ 10s | 98℃ 10s |
   | 62℃ 8s | 61℃ 8s |
   | 72℃ 18s | 72℃ 19s |
   | 72℃ 3min | 72℃ 3min |
   | 12℃ ∞ | 12℃ ∞ |
   | Final concentration (after purification) | 8.1ng/μl | 14.9ng/μl |

   Gel analyses and purification/gel extraction.

![](https://static.igem.org/mediawiki/2015/d/de/SJTUB_0718%EF%BC%883%EF%BC%89.png)

The final concentration is not satisfying enough because the extra band existed and gel extraction had to be operated. We adjusted several conditions and tried for a couple of days. During this time, we also changed restriction reaction site and redesigned the primers. See the final version of the protocol of these two fragments at “20150729”.

1. PCR for fragments Ecolivk and sll1338 (both are signal sequences) from the synthetic plasmid. But we failed all the time. The main problems are: the concentration of the PCR products are low and for the fragments are too small (both under 120bp) to stay stably on the column when purifying, the purified products could not be used for downstream operation. We tried and adjusted constantly for at least half a month, but no satisfying results were showed.

### [![SJTUB transformation logo.jpg](/wiki/images/3/31/SJTUB_transformation_logo.jpg)](/File%3ASJTUB_transformation_logo.jpg) Transformation

We used plasmid which contains ADS as our positive control to learn the whole process of transformation.

### [![SJTUB testing logo.jpg](/wiki/images/6/66/SJTUB_testing_logo.jpg)](/File%3ASJTUB_testing_logo.jpg) Testing

## Week 3 (July 20 ~)

### [![SJTUB hp logo.jpg](/wiki/images/7/79/SJTUB_hp_logo.jpg)](/File%3ASJTUB_hp_logo.jpg) Human Practice

We reorganized the new plan and decided to do our Human Practice project work on the line of the process flow we planned for the desalination, including intake, bio-desalination itself, cell-water separation and the steps after. We can have like a subtopic for each step. In addition, it will be the communication with other iGEM teams and public acceptance.

### [![SJTUB construction logo.jpg](/wiki/images/c/c3/SJTUB_construction_logo.jpg)](/File%3ASJTUB_construction_logo.jpg) Construction

#### July 21

1. Try different conditions of overlapping PCR for the whole construction of P\_dark-HR\* and pCPCG2-HR\*.
2. The final concentration of digestion product was too low for downstream operation. We suspected that it might be misoperation. Also we found out that we used isocaudarners XbaI and BcuI (also called SpeI) as digestion reaction sites, so we changed the two sites later.

#### July 22

1. Try different conditions of overlapping PCR for the whole construction of P\_dark-HR\* and pCPCG2-HR\*.
2. Restriction enzyme digestion of pBluescript. CIAP was added in one sample and another one was not. Moreover, the reaction time was lengthened. We found out that CIAP didn’t seem to be that useful in our experiment. But longer reaction time was indeed needed.

#### July 23

1. Try different conditions of overlapping PCR for the whole construction of P\_dark-HR\* and pCPCG2-HR\*.
2. We decided to make sure that which method of plasmid‘s digestion reaction is better, single digest or double digest and which enaturation way is better. So we designed several controls using the wrong enzymes as a precursive test. It’s also because that we need to purchase the new enzyme at the time.

   | Digestion reaction | pBluescript(D) | pBluescript(S1) | pBluescript(S2) | P\_dark-HR\*/ pCPCG2-HR\* |
   | --- | --- | --- | --- | --- |
   | ddwater | 9μl | 9μl | 9μl | Fill up to 30μl |
   | 10×（green）buffer | 2μl | 2μl | 2μl | 2μl |
   | Plasmid/DNA fragment | 7.2μl(1μg) | 7.2μl(1μg) | 7.2μl(1μg) | 1μg |
   | BcuI | 1μl | 1μl | 1μl | 1μl |
   | XbaI | 1μl | 1μl | 1μl | 1μl |
   | Total volume | 20μl | 20μl | 20μl | 30μl |
   |
   | Reaction | 37℃ for 20mins | 37℃ for 20mins | 37℃ for 20mins |  |
   | Denaturation | Gel extraction | Purification | Thermodynamic denauration |  |

   | Ligation | P\_dark-HR\*(D)/ pCPCG2-HR\*(D) | P\_dark-HR\*(S1)/ pCPCG2-HR\*(S1) | P\_dark-HR\*(S2)/ pCPCG2-HR\*(S2) |
   | --- | --- | --- | --- |
   | ddwater | Fill up to 10μl | Fill up to 10μl | Fill up to 10μl |
   | 10×buffer | 1μl | 1μl | 1μl |
   | Vector DNA | 60ng | 60ng | 60ng |
   | Insert DNA | 300ng | 300ng | 300ng |
   | Ligase | 0.2μl(1WeissU) | 0.2μl | 0.2μl |
   | Total volume | 10μl | 10μl | 10μl |
   |
   | Reaction | 17℃ for 4h | 17℃ for 4h | 17℃ for 4h |
   | Denaturation | Purification/None | Purification/None | Purification/None |

   Transformation.
   Results:

   | pCPCG2-HR(D) | pCPCG2-HR(S1) | pCPCG2-HR(S2) |
   | --- | --- | --- |
   | Normal | Normal | Very few colonies |
   | P\_dark-HR(D) | P\_dark-HR(S1) | P\_dark-HR(S2) |
   | Normal | No colony | Very few colonies |

### [![SJTUB transformation logo.jpg](/wiki/images/3/31/SJTUB_transformation_logo.jpg)](/File%3ASJTUB_transformation_logo.jpg) Transformation

The natural transformation includes transformation, solid segregation, liquid segregation, the whole process took about three weeks.

### [![SJTUB testing logo.jpg](/wiki/images/6/66/SJTUB_testing_logo.jpg)](/File%3ASJTUB_testing_logo.jpg) Testing

## Week 4 (July 27 ~)

### [![SJTUB hp logo.jpg](/wiki/images/7/79/SJTUB_hp_logo.jpg)](/File%3ASJTUB_hp_logo.jpg) Human Practice

We finished some of our work in public acceptance. We found out that there has been data about how short the water is in China, but they are cold charts. Therefore, we made it into data maps so everyone can understand it.

We also did some research in cell-water separation and toxin, especially toxin. There was a few kind of major toxin, but not in our strain gene.

### [![SJTUB drylab logo.jpg](/wiki/images/4/41/SJTUB_drylab_logo.jpg)](/File%3ASJTUB_drylab_logo.jpg) Modeling

Learn how to use modeling tools both mathematical tools like ODEs and PDEs, and software like MATLAB.

### [![SJTUB construction logo.jpg](/wiki/images/c/c3/SJTUB_construction_logo.jpg)](/File%3ASJTUB_construction_logo.jpg) Construction

#### July 29

1. Overlapping PCR for the whole construction of P\_dark-HR\* and pCPCG2-HR\* using new primers in order to add new restriction enzyme reaction sites XbaI and PstI.

   |  | P\_dark-HR\* | pCPCG2-HR\* |
   | --- | --- | --- |
   | ddwater | 88μl | 88μl |
   | Template1 | P\_dark-HR-PcpcB 1.3μl(49.7ng/50μl) | pCPCG2-HR-PcpcB 1.4μl(52.8ng/50μl) |
   | Template2 | PcpcB-Kn 2.9μl(55.7ng/50μl) | PcpcB -Kn 2.9μl(55.7ng/50μl) |
   | Primer1 | 4μl | 4μl |
   | Primer2 | 4μl | 4μl |
   | PrimeSTAR Mix | 100μl | 100μl |
   | Total volume | 200μl(25μl\*8) | 200μl(25μl\*8) |
   |
   | 2-step PCR （round 1）×8 cycle | 98℃ 3min | |
   | 98℃ 10s | |
   | 72℃ 1min15s | |
   | 72℃ 3min | |
   | 12℃ ∞ | |
   | 2-step PCR (round2) ×33 cycle | 98℃ 3min | |
   | 98℃ 10s | |
   | 72℃ 1min50s | |
   | 72℃ 3min | |
   | 12℃ ∞ | |
   | Final concentration (after purification) | 72.4ng/μl | 67.7ng/μl |

   Gel analyses and purification/gel extraction.

![](https://static.igem.org/mediawiki/2015/3/35/SJTUB_0729.png)

#### July 30

1. Restriction reaction and ligation reaction of pBluescript and DNA fragments P\_dark-HR\*, pCPCG2-HR\* with XbaI and PstI.

   | Digestion reaction | pBluescript | P\_dark-HR\* | pCPCG2-HR\* |
   | --- | --- | --- | --- |
   | ddwater | 45μl | 86.8μl | 90μl |
   | 10×(green) buffer | 10μl | 8μl | 8μl |
   | Plasmid/DNA fragment | 35μl(1μg) | 17.2μl(1μg) | 14μl |
   | XbaI | 5μl | 4μl | 4μl |
   | PstI | 5μl | 4μl | 4μl |
   | Total volume | 100μl(20μl\*5) | 120μl(30μl\*4) | 120μl(30μl\*4) |
   |
   | Reaction | 37℃ for 20min | 37℃ for 1h | 37℃ for 1h |
   | Denaturation | Gel extraction | Purification | Purification |

   | Ligation reaction | P\_dark-HR\*-pBluescript | pCPCG2-HR\*-pBluescript |
   | --- | --- | --- |
   | ddwater | 2.8μl | 2.8μl |
   | 10×buffer | 1μl | 1μl |
   | Vector DNA | 1μl(72.1ng) | 1μl(72.1ng) |
   | Insert DNA | 5μl(362ng) | 5μl(338.5ng) |
   | Ligase | 0.3μl | 0.3μl |
   | Total volume | 10μl | 10μl |
   |
   | Reaction | 17℃ for 4h | 17℃ for 4h |
   | Denaturation | Thermodynamic | Thermodynamic |
2. Transformation of P\_dark-HR\*-pBluescript and pCPCG2-HR\*-pBluescript.

#### July 31

1. Inoculation: 16 samples for each construction.

#### Aug. 1

1. Bacterial liquid PCR.

   |  |  |
   | --- | --- |
   | Forward primer | 1μl (10μM) |
   | Reverse primer | 1μl (10μM) |
   | Template(bacterial liquid) | 1μl |
   | 2×Taq Mix | 10μl |
   | diwater | Fill up to 20μl |
   | Total volume | 20μl |
   |
   | ×25 cycle | 95℃ 3min |
   | 94℃ 15s |
   | 68℃ 15s |
   | 72℃ 3min50s |
   | 72℃ 5min |
   | 12℃ ∞ |

   Gel analyses.

![](https://static.igem.org/mediawiki/2015/e/e6/SJTUB_0801.png)

1. Plasmid extraction.
2. Restriction enzyme reaction examination.

![](https://static.igem.org/mediawiki/2015/8/85/SJTUB_0801%282%29.png)

1. Transformation of pCPCG2-HR\*-pBluescript, using blue-white screen.
2. Prepare 10× TBE mother liquor.
3. Sequence the examined plasmid containing construction P\_dark-HR\*-pBluescript.

#### Aug. 2

1. Inoculation: 17 samples.
2. Bacterial liquid PCR.

![](https://static.igem.org/mediawiki/2015/7/76/SJTUB_0802.png)

### [![SJTUB transformation logo.jpg](/wiki/images/3/31/SJTUB_transformation_logo.jpg)](/File%3ASJTUB_transformation_logo.jpg) Transformation

The eletro-transformation also includes electrotransformation, solid segregation and liquid segregation, but the whole process took almost four weeks.

We tried the eclctrotransformation of pdark-HR but failed.

### [![SJTUB testing logo.jpg](/wiki/images/6/66/SJTUB_testing_logo.jpg)](/File%3ASJTUB_testing_logo.jpg) Testing

## Week 5 (August 3 ~)

### [![SJTUB hp logo.jpg](/wiki/images/7/79/SJTUB_hp_logo.jpg)](/File%3ASJTUB_hp_logo.jpg) Human Practice

We went to Hangzhou Water Treatment Technology Development Center. We had send some of our questions before we arrived about desalination industry and technology. They planned a presentation that is very useful. An important change is instead of thinking replacing those desalination method, we found a new idea to cooperate with them.

### [![SJTUB construction logo.jpg](/wiki/images/c/c3/SJTUB_construction_logo.jpg)](/File%3ASJTUB_construction_logo.jpg) Construction

#### Aug. 3

1. Plasmid extraction
2. Restriction enzyme reaction examination.

![](https://static.igem.org/mediawiki/2015/0/0e/SJTUB_0803.png)

#### Aug. 5

1. Propagating cell culture of P\_dark-HR\*-pBluescript and cell conservation.

#### Aug. 6

1. Plasmid extraction.

### [![SJTUB transformation logo.jpg](/wiki/images/3/31/SJTUB_transformation_logo.jpg)](/File%3ASJTUB_transformation_logo.jpg) Transformation

#### pdark natural transformation

##### In the noon of Aug. 5

Inoculated wildtype cyanobacteria into the flask and make sure the initial OD was about 0.1;

##### In the evening of Aug. 7

When the OD of our culture reached 0.4-0.6, collect the cell and mix the DNA, cyanobacteria and liquid medium, meanwhile set a negative control culture (without DNA), put them into illumination incubator at 30℃;

##### Aug. 7

Shook by hand for about 4 times from morning（every 2-3 hours）, after 8 hours, took 200ul culture of each tube, spread them on different solid plates which contained 20 micrograms kanamycin per milliliter, put the plate into illumination incubator at 30℃.

#### pcpcG2 electro-transformation

##### At the noon of Aug. 8

We inoculated wildtype cyanobacteria into the flask and make sure the initial OD was about 0.1; prepare the electric tubes

### [![SJTUB testing logo.jpg](/wiki/images/6/66/SJTUB_testing_logo.jpg)](/File%3ASJTUB_testing_logo.jpg) Testing

## Week 6 (August 10 ~)

### [![SJTUB hp logo.jpg](/wiki/images/7/79/SJTUB_hp_logo.jpg)](/File%3ASJTUB_hp_logo.jpg) Human Practice

We brought some of our work in public acceptance to Shanghai Science and Technology Museum. This presentation made us want to do it more prepared to a bigger audience.

### [![SJTUB drylab logo.jpg](/wiki/images/4/41/SJTUB_drylab_logo.jpg)](/File%3ASJTUB_drylab_logo.jpg) Modeling

Reviewing literature and get ready for modeling

### [![SJTUB construction logo.jpg](/wiki/images/c/c3/SJTUB_construction_logo.jpg)](/File%3ASJTUB_construction_logo.jpg) Construction

#### Aug. 10

1. Prepare 34mg/ml chloramphenicol.
2. Propagating cell culture of pCPCG2-HR\*-pBluescript and cell conservation.

#### Aug. 11

1. Plasmid extraction.

#### Aug. 12

1. Transformation of BBa\_J04450.

#### Aug. 13

1. Inoculation: 10 samples.

#### Aug. 14

1. Plasmid extraction.
2. Linearize plasmid by restriction enzyme reaction. Extract the linearized plasmid.

#### Aug. 16

1. Inoculation of DH5α for component cell making.

### [![SJTUB transformation logo.jpg](/wiki/images/3/31/SJTUB_transformation_logo.jpg)](/File%3ASJTUB_transformation_logo.jpg) Transformation

#### pcpcG2 electro-transformation

##### In the evening of Aug. 10

Put electric tubes into 60℃ dryer for at least 2 hours, sterilized them by UV before electrotransformation.

Collect the cyanobacteria cell and diluted the cell with sterilized ddH2O washed it for 5 times,then diluted it with sterile ddH2O, mixed with pcpcg2-HR plasmid, transfered them into the electric tube, tunk for several times so that the liquid can reach to the bottom, installed the tube into switch, did electro-transformation, then transfer the mixture to BG11 culture

##### In the evening of Aug. 11

Centrifuged the cyanobacteria, diluted the cell with 400ul liquid culture, mixed with 5ml soft agar BG11 medium, spread on solid medium which contained 20 micrograms kanamycin per milliliter, put the plate into illumination incubator at 30 ℃.

### [![SJTUB testing logo.jpg](/wiki/images/6/66/SJTUB_testing_logo.jpg)](/File%3ASJTUB_testing_logo.jpg) Testing

## Week 7 (August 17 ~)

### [![SJTUB hp logo.jpg](/wiki/images/7/79/SJTUB_hp_logo.jpg)](/File%3ASJTUB_hp_logo.jpg) Human Practice

We went to Shanghai Water Supply Authority; it is a supplement visit for something we write. It is good to verify what you write to reality.

### [![SJTUB construction logo.jpg](/wiki/images/c/c3/SJTUB_construction_logo.jpg)](/File%3ASJTUB_construction_logo.jpg) Construction

#### Aug. 17

1. Component cell making.

#### Aug. 20

1. Producted linearized Pdark-GFP, amplified GFP fragments from constructed plasmid GFP.
2. Agarose gel electrophoresis for PCR products.
3. Extracted positive plasmids (16 tubes).
4. Inoculated 14 D3 colonies and negative control C2/D2 for 2 tubes. (16 tubes in total)

#### Aug. 21

1. Extracted plasmids from the 16 tubes inoculated on 8.20.
2. Agarose gel electrophoresis test and restriction enzyme digestion test for plasmids above.(20μl system included 1μl SacI and 1μl EcoRI.)
3. Colony PCR test for D3 and C2/D2.
4. Prepared homologous recombination system (as is shown below).
5. Transformation and plating afterwards.

|  | GFP-1\* | GFP-2\* | GFP-3a | GFP-3b |
| --- | --- | --- | --- | --- |
| ddwater | 12μl | 11μl | 10μl | 9μl |
| 5x CEII Buffer | 4μl | 4μl | 4μl | 4μl |
| Linearized plasmid Pdark-HR(-GFP-)-1 | 3μl | 3μl | 3μl | 3μl |
| GFP fragment | 1μl | 0μl | 1μl | 2μl |
| Exnase II | 0μl( | 2μl | 2μl | 2μl |

* \*GFP-1&2 are 2 control groups.

#### Aug. 22

Colony PCR for recombination cells. 12tubes for single colonies of GFP 3a and 3b, 3 tubes for control groups GFP-1 and GFP-2.

|  | GFP-3a/3b/1/2 |
| --- | --- |
| ddwater | 144μl |
| Template | 0.5μl(each tube) |
| Primer M13F(10μM) | 8μl |
| Primer GFP-R(10μM) | 8μl |
| 2xTaq Mix | 160μl |
| Total volume | 320μl(20μl\*16) |

[![SJTUB 0822.jpg](/wiki/images/4/42/SJTUB_0822.jpg)](/File%3ASJTUB_0822.jpg)

As is shown in the figure, product no.6 showed negative and control no.1 showed positive.
Most of the products qualified the length of 1400bp or so.

#### Aug. 23

Mass prep of Pdark-GFP plasmid (No.1,4).

### [![SJTUB transformation logo.jpg](/wiki/images/3/31/SJTUB_transformation_logo.jpg)](/File%3ASJTUB_transformation_logo.jpg) Transformation

#### pdark natural transformation

##### In the evening of Aug 18

We picked 4 colonies (marked with 1,2,3 & 4) from the pdark-HR plate and spread them on another BG11 plate in parallel seprately to do solid segregation

##### In the evening of Aug. 20

We scraped the grown cell from the plate, which is 1,3 & 4 to do colony PCR, 1st liquid segregation and solid storage

#### pre-experiment of pdark-HR

##### In the evening of Aug. 20

Inoculated 16 wildtype & 16 pdark-HR (use pdark-HR from electro-transformation), added 11g/L or 22g/L NaCl & retinene at the beginning

##### In the evening of Aug. 22

Induced with darkness, took the first 4 samples at 9pm

##### In the evening of Aug. 23

Cultivated with white light, took the second 4 samples at 9pm.

### [![SJTUB testing logo.jpg](/wiki/images/6/66/SJTUB_testing_logo.jpg)](/File%3ASJTUB_testing_logo.jpg) Testing

## Week 8 (August 24 ~)

### [![SJTUB hp logo.jpg](/wiki/images/7/79/SJTUB_hp_logo.jpg)](/File%3ASJTUB_hp_logo.jpg) Human Practice

We struggled to come up with the report of intake. We planned to do collaboration, yet, we want something that will cover the necessity of this project. At last, we decide to add a section to explain the trend of water supply and the estimated trend of desalination resource water. This will provide us a stronger why.

### [![SJTUB drylab logo.jpg](/wiki/images/4/41/SJTUB_drylab_logo.jpg)](/File%3ASJTUB_drylab_logo.jpg) Modeling

Determine the main idea about modeling

### [![SJTUB construction logo.jpg](/wiki/images/c/c3/SJTUB_construction_logo.jpg)](/File%3ASJTUB_construction_logo.jpg) Construction

#### Aug. 25

PCR cloning of fragment Pdark-GFP and gel extraction.
Final concentration: 119.5ng/μl (tube1); 304ng/μl (tube2)

#### Aug. 26

Linearization of pSB1C3 plasmid by PCR.

|  | BBa\_J0450 |
| --- | --- |
| ddwater | 160μl |
| Template | 32μl |
| Primer pSB-F(10μM) | 8μl |
| Primer pSB-R(10μM) | 8μl |
| PrimeSTAR Mix | 200μl |
| Total volume | 400μl(50μl\*8) |

DpnI digestion for 2h. Gel extraction.

[![SJTUB 0826.jpg](/wiki/images/c/c4/SJTUB_0826.jpg)](/File%3ASJTUB_0826.jpg)

Shipping part Pdark-GFP, Pdark-HR and PcpcG2-HR into pSB1C3 plasmid via PCR - Homologous recombination method (24 tubes).

|  | Parts | Control-1(\*3) | Control-2(\*3) |
| --- | --- | --- | --- |
| ddwater | 300μl | 14μl | 15μl |
| 5x CEII Buffer | 100μl | 4μl | 4μl |
| Linearized plasmid pSB1C3 | 25μl | 1μl | 0μl |
| Fragment (Pdark-GFP/HR,PcpcG2-HR) | 25μl | 1μl | 1μl |
| Exnase II | 50μl | 0μl | 2μl |

Agarose Gel electrophoresis test.

#### Aug. 27

Transformation into DH5a (4 tubes not including no-fragment control) and plating afterwards.

```
* (Lack of control group led to a failure of this transformation.)
```

[![SJTUB 0827.jpg](/wiki/images/8/89/SJTUB_0827.jpg)](/File%3ASJTUB_0827.jpg)

Test with colony PCR and restriction enzyme reaction (XbaI and PstI).

Plasmid extraction (Pdark-GFP).

#### Aug. 28

Linearization of official vector plasmid pSB1C3.

|  | BBa\_J0450 |
| --- | --- |
| ddwater | 160μl |
| Template | 32μl |
| Primer pSB-F(10μM) | 8μl |
| Primer pSB-R(10μM) | 8μl |
| PrimeSTAR Mix | 200μl |
| Total volume | 400μl(50μl\*8) |

DpnI digestion for 2h. Gel extraction.

PCR for fragment PcpcG2-HR.

|  | PcpcG2-HR | Pdark-HR |
| --- | --- | --- |
| ddwater | 220μl | 220μl |
| Template | 10μl | 10μl |
| Primer UHKD-F(10μM) | 10μl | 10μl |
| Primer UHKD-R(10μM) | 10μl | 10μl |
| PrimeSTAR Mix | 250μl | 250μl |
| Total volume | 500μl(50μl\*8) | 500μl(50μl\*8) |

Agarose gel electrophoresis test and gel extraction for Pdark-HR and PcpcG2-HR fragment.

Shipping part Pdark-HR and PcpcG2-HR into pSB1C3 plasmid via PCR - Homologous recombination method.

Second try of homologous recombination.

|  | Pdark-HR | PcpcG2-HR | Non-fragment control |
| --- | --- | --- | --- |
| ddwater | 12μl | 12μl | 13μl |
| 5x CEII Buffer | 4μl | 4μl | 4μl |
| Linearized plasmid pSB1C3 (80ng/μl) | 1μl | 1μl | 1μl |
| Fragment | 1μl | 1μl | 0μl |
| Exnase II | 2μl | 2μl | 2μl |

Transformation into DH5a (3 tubes) and plating afterwards.

Spread plates and cultured for 14h in 37℃.

#### Aug. 29

Inoculation of Pdark-GFP, Pdark-HR and PcpcG2-HR transformed DH5a. (11 tubes each, 3 from control group)

[![SJTUB 0829.jpg](/wiki/images/3/3b/SJTUB_0829.jpg)](/File%3ASJTUB_0829.jpg)

#### Aug. 30

Bacteria Liquid PCR fpr Pdark-GFP, Pdark-HR and PcpcG2-HR.
Agarose gel electrophoresis test.

Pdark-GFP:

|  |  |
| --- | --- |
| pSB-vF | 1μl (10μM) |
| GFP-R | 1μl (10μM) |
| Bacterial liquid(Pdark-GFP) | 1μl |
| 2×Taq Mix | 10μl |
| ddwater | Fill up to 20μl |
| Total volume | 20μl |
|
| 95℃ 3min |  |
| 94℃ 15s | ×28 cycle |
| 48℃ 15s |
| 72℃ 1min20s |
| 72℃ 5min |  |
| 12℃ ∞ |  |

Pdark-HR:

|  |  |
| --- | --- |
| pSB-vF | 1μl (10μM) |
| SS-HR-R | 1μl (10μM) |
| Bacterial liquid(Pdark-GFP) | 1μl |
| 2×Taq Mix | 10μl |
| ddwater | Fill up to 20μl |
| Total volume | 20μl |
|
| 95℃ 3min |  |
| 94℃ 15s | ×28 cycle |
| 48℃ 15s |
| 72℃ 50s |
| 72℃ 5min |  |
| 12℃ ∞ |  |

PcpcG2-HR:

|  |  |
| --- | --- |
| pSB-vF | 1μl (10μM) |
| SS-HR-R | 1μl (10μM) |
| Bacterial liquid(Pdark-GFP) | 1μl |
| 2×Taq Mix | 10μl |
| ddwater | Fill up to 20μl |
| Total volume | 20μl |
|
| 95℃ 3min |  |
| 94℃ 15s | ×28 cycle |
| 48℃ 15s |
| 72℃ 60s |
| 72℃ 5min |  |
| 12℃ ∞ |  |

### [![SJTUB transformation logo.jpg](/wiki/images/3/31/SJTUB_transformation_logo.jpg)](/File%3ASJTUB_transformation_logo.jpg) Transformation

#### pdark natural transformation

##### In the evening of Aug. 29

2nd liquid segregation of pdark-HR

#### pcpcG2 electro-transformation

##### In the evening of Aug. 24

We picked 4 colonies (marked with 1,2,3 & 4) from the pcpcG2-HR plate and spread them on another BG11 plate in parallel seprately to do solid segregation

##### In the evening of Aug. 30

We scraped the grown cell from the plate, which is 2,3 & 4 to do colony PCR, 1st liquid segregation and solid storage

#### pre-experiment of pdark-HR

##### Aug. 24

Took the third 4 samples at 9am & fourth 4 samples at 9pm.

##### Aug. 25

Took the fifth 4 samples at 9am & sixth 4 samples at 9pm.

##### Aug. 26

Took the seventh 4 samples at 9am & eighth 4 samples at 9pm.

#### pdark-GFP electro-transformation

##### In the evening of Aug. 24

Set culture.

##### In the evening of Aug. 26

Electro-transformation of pdark-GFP.

##### In the evening of Aug. 27

Collected half of the cell and mixed with 5ml soft agar, then poured on solid medium plate.

### [![SJTUB testing logo.jpg](/wiki/images/6/66/SJTUB_testing_logo.jpg)](/File%3ASJTUB_testing_logo.jpg) Testing

## Week 9 (August 31 ~)

### [![SJTUB hp logo.jpg](/wiki/images/7/79/SJTUB_hp_logo.jpg)](/File%3ASJTUB_hp_logo.jpg) Human Practice

We worked on the desalination part. Most of the information in this section we already knew, so, it is an easy week.

### [![SJTUB drylab logo.jpg](/wiki/images/4/41/SJTUB_drylab_logo.jpg)](/File%3ASJTUB_drylab_logo.jpg) Modeling

Construct the dynamic system model using ODE

### [![SJTUB construction logo.jpg](/wiki/images/c/c3/SJTUB_construction_logo.jpg)](/File%3ASJTUB_construction_logo.jpg) Construction

### Aug. 31

1. Plasmid extraction.
2. Double restriction enzyme digestion test for Pdark-GFP, Pdark-HR and PcpcG2-HR.

|  | Restriction Enzyme Digestion |
| --- | --- |
| ddwater | 360μl |
| FD buffer | 450μl |
| XbaI | 45μl |
| PstI | 45μl |
| Template | 1μl/tube |
| Total volume | 900μl(20μl\*39) |

Agarose gel extraction test.

### Sep. 2

Gel anlysis of the restriction enzyme digestion product for a second time.

### Sep. 3

Sequencing samples. Pdark-GFP-3, Pdark-GFP-8, PcpcG2-HR-4,PcpcG2-HR-6, Pdark-HR-2, Pdark-HR-4.

### Sep. 6

All sequenced sample were successful and right.
Site-directed mutation on PcpcG2-HR to eliminate the EcoRI sites via PCR method.

|  |  |
| --- | --- |
|  | PcpcG2-HR-Mut |
| ddwater | 105μl |
| G2Mut-F | 10μl (10μM) |
| G2Mut-R | 10μl (10μM) |
| PcpcG2-HR(pSB1C3) | 2μl |
| PrimeStar Mix | 125μl |
| Total volume | 250μl（25μl\*8）( |
|
| 98℃ 3min |  |
| 98℃ 10s | ×32 cycle |
| 65℃ 5s |
| 72℃ 30s |
| 72℃ 5min |  |
| 12℃ ∞ |  |

DpnI digestion for 2h. Electrophoresis test and gel extraction afterwards.

Homologous recombination of PcpcG2-HR-Mut.

|  | PG2-Mut | Control-1 |
| --- | --- | --- |
| ddwater | 12μl | 14μl |
| 5x CEII Buffer | 4μl | 4μl |
| PCR product | 2μl | 2μl |
| Exnase II | 2μl | 0μl |

37℃ for 30min.
Product storaged in -20℃ overnight.

### [![SJTUB transformation logo.jpg](/wiki/images/3/31/SJTUB_transformation_logo.jpg)](/File%3ASJTUB_transformation_logo.jpg) Transformation

#### pdark natural transformation

##### In the evening of Aug. 31

3rd liquid segregation of pdark-HR

##### In the evening of Sep. 3

4th liquid segregation of pdark-HR

##### In the evening of Sep. 6

5th liquid segregation of pdark-HR

#### pcpcG2 electro-transformation

##### In the evening of Sep. 2

2nd liquid segregation of pcpcG2-HR

##### In the evening of Sep. 4

3rd liquid segregation of pcpcG2-HR

##### In the evening of Sep. 6

4th liquid segregation of pcpcG2-HR

### [![SJTUB testing logo.jpg](/wiki/images/6/66/SJTUB_testing_logo.jpg)](/File%3ASJTUB_testing_logo.jpg) Testing

#### second desalination test of pdark-HR

##### Sep. 2

inoculated 24 wildtype & 24 pdark-HR (use pdark-HR from colony 3) at 9pm

##### Sep. 4

induced with darkness at 9pm, added 22g/L NaCl & retinene, then took the first 6 samples (3 parallel samples for each)

##### Sep. 5

cultivated with white light at 9am, then took the second 6 samples,

took the third 6 samples at 10am

took the fourth 6 samples at 11am

took the fifth 6 samples at 1pm

took the sixth 6 samples at 9pm

##### Sep. 6

took the seventh 6 samples at 9am

took the eighth 6 samples at 9pm

## Week 10 (September 7 ~)

### [![SJTUB hp logo.jpg](/wiki/images/7/79/SJTUB_hp_logo.jpg)](/File%3ASJTUB_hp_logo.jpg) Human Practice

Connecting everything together.

### [![SJTUB drylab logo.jpg](/wiki/images/4/41/SJTUB_drylab_logo.jpg)](/File%3ASJTUB_drylab_logo.jpg) Modeling

Coding and simulating using MATLAB and plot the result

### [![SJTUB construction logo.jpg](/wiki/images/c/c3/SJTUB_construction_logo.jpg)](/File%3ASJTUB_construction_logo.jpg) Construction

### Sep. 7

Transformation of PcpcG2-HR-Mut into DH5a and plating.
After incubation for 12h,
Inoculated single colonies into liquid LB culture (10 tubes for test group and 2 for control group).

### Sep. 8

Plasmid extraction.

Double restriction enzyme digestion.

|  | Restriction Enzyme Digestion |
| --- | --- |
| ddwater | 280μl |
| FD buffer | 40μl |
| XbaI | 20μl |
| EcoRI | 20μl |
| Template | 2μl/tube |
| Total volume | 400μl(20μl\*20) |

* Template included PcpcG2-HR-Mut, PcpcG2-HR and 0 control.

Agarose gel electrophoresis test.

### Sep. 9

Sequencing PcpcG2-HR-Mut-4 and PcpcG2-HR-Mut-9.

### Sep. 10

Sequencing results proved right.

### Sep. 17

Functional fragments restriction enzyme digestion.

|  | Pdark-GFP | Pdark-HR | PcpcG2-HR |
| --- | --- | --- | --- |
| ddwater | 15μl | 15μl | 15μl |
| FD buffer | 2μl | 2μl | 2μl |
| XbaI | 1μl | 1μl | 1μl |
| PstI | 1μl | 1μl | 1μl |
| Template | 1.3μl | 1μl | 0.9μl |
| Total volume | 20μl | 20μl | 20μl |

[![SJTUB 0917.jpg](/wiki/images/f/fd/SJTUB_0917.jpg)](/File%3ASJTUB_0917.jpg)

### [![SJTUB transformation logo.jpg](/wiki/images/3/31/SJTUB_transformation_logo.jpg)](/File%3ASJTUB_transformation_logo.jpg) Transformation

#### pdark natural transformation

##### In the evening of Sep. 11

6th liquid segregation of pdark-HR

#### pcpcG2 electro-transformation

##### In the evening of Sep. 11

5th liquid segregation of pcpcG2-HR

#### pdark-GFP electro-transformation

##### Sep. 8

We picked 1 colony (only 1 colony appeared) from the pdark-GFP plate and spread them on another BG11 plate in line to do solid segregation

### [![SJTUB testing logo.jpg](/wiki/images/6/66/SJTUB_testing_logo.jpg)](/File%3ASJTUB_testing_logo.jpg) Testing

#### first desalination test of pcpcG2-HR

##### Sep. 7

Inoculated 15 wildtype & 15 pcpcG2-HR (use pcpcG2-HR from colony 2) at 10pm.

##### Sep. 9

Induced with green light at 10pm, added 22g/L NaCl & retinene, then took the first 6 samples (3 parallel samples for each).

##### Sep. 10

Cultivated with red light at 10am, then took the second 6 samples,

Took the third 6 samples at 2pm.

Took the fourth 6 samples at 10pm.

##### Sep. 11

Took the fifth 6 samples at 10am.

#### Detection of Membrane Localization of Halorhodopsin by Immunocytochemistry

To examine whether the HR expressed successfully onto the cell membrane, we designed an immunocytochemical experiment. For that the C terminal of the HR sequence we designed contains a His-tag, we used anti-His mAb as the primary antibody and goat anti-mouse IgG Alexa Fluor 488(green fluorescence) as the second antibody. And if a loop of green fluorescence can be seen under the confocal microscope, we will ensure that the HR expressed successfully onto the cell membrane.
A few things should be paid attention to:

1. The incubating condition can be overnight at 4℃ or 2 hours at 37℃. But if a better result is wanted, use the former condition.
2. Be careful that the procedures involving fluorescent antibodies need to be operated under lucifugal condition.

##### Sep. 15

1ml cyanobacteria liquid for each sample was prepared, including four experiment groups--Pdark-HR(green), Pdark-HR(red), PcpcG2-HR(green) and PcpcG2-HR(red), which had been cultured to OD730=0.6 and induced for 12h, and controls—wildtype(green) and wildtype(red), which were not been induced.

Anti-His mAb was applied with PBS in proportion of 1:100 as the primary antibody. After 2 hours’ incubating(at 37℃) and washing steps, goat anti-mouse IgG Alexa Fluor 488(green fluorescence) and goat anti-mouse IgG Alexa Fluor 594(red fluorescence) were applied respectively with PBS in proportion of 1:1000 as the second antibodies.

After 2 hours’ incubating(at 37℃) and washing steps, the cyanobacteria pellets were resuspended with 50μl PBS. Six slides were prepared. While observing under a confocal microscope, we found out that the cyanobacteria has spontaneous red fluorescence. Several positive results can be seen but the signal is not strong enough. So we decided to optimize the protocol and redo it.

##### Sep.15 evening to Sep. 16

1ml cyanobacteria liquid for each sample was prepared, including four experiment groups--Pdark-HR(1:100), Pdark-HR(1:50), PcpcG2-HR(1:100) and PcpcG2-HR(1:50), which had been cultured to OD730=0.6 and induced for 16h, and controls—wildtype(1:100) and wildtype(1:50), which had been induced for 16h as well.

The primary antibody anti-His mAb was applied with PBS in proportion of 1:100 or 1:50 as mentioned. The samples were incubated at 4℃ overnight. The second antibody goat anti-mouse IgG Alexa Fluor 488(green fluorescence) was applied with PBS in proportion of 1:300 and the samples were incubated at 37℃ for 2 hours.

After washing the samples, the cyanobacteria pellets were resuspended with 20μl PBS. Six slides were prepared. The results were better than the former one. Concerning of time, we only got the micrographs of the samples containing the primary antibody with the concentration of 1:50.

[![SJTUB 5130809056.png](/wiki/images/2/21/SJTUB_5130809056.png)](/File%3ASJTUB_5130809056.png)

In the experiment groups (Pdark-HR and PcpcG2-HR), many loops of green fluorescence can be seen under the confocal microscope while the control groups (wildtype) cannot, which indicates that the halorhodopsin expressed successfully on the cell membrane of the genetic cyanobacteria instead of any other position in the cells.

Bio-X Institutes

Shanghai Jiao Tong University

800, Dongchuan Road 200240, Shanghai, China

kariny888@sjtu.edu.cn

[![](https://static.igem.org/mediawiki/2015/0/03/SJTUB_SJTU_logo.png)](http://www.sjtu.edu.cn/ "nima")
[![](https://static.igem.org/mediawiki/2015/9/95/SJTUB_biox_logo.png)](http://www.bio-x.cn/)
[![](https://static.igem.org/mediawiki/2015/f/f3/SJTUB_igemfoundation.png)](http://www.sjtu.edu.cn/)

[![](https://static.igem.org/mediawiki/2015/2/24/SJTUB_Merk_logo.png)](http://www.merck.com/index.html)
[![](https://static.igem.org/mediawiki/2015/e/e4/SJTUB_genwiz_logo.png)](http://www.genewiz.com/)
[![](https://static.igem.org/mediawiki/2015/0/08/SJTUB_life_logo.png)](http://life.sjtu.edu.cn/)