iGEM 2017 — Team CIEI-China

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# Team:CIEI-China/Notebook

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* [Jan. 2017](#a1)* [Mar. 2017](#a2)* [Apr.2017](#a3)* [May. 2017](#a4)* [Jun. 2017](#a5)* [Week 1-2 in Jul. 2017](#a6)* [Week 3 in Jul. 2017](#a7)* [Week 4 in Jul. 2017 to week 1 in Aug. 2017](#a8)* [Week 2 –week 3 in Aug.2017](#a9)* [Week 4 in Aug. 2017 to week 1 in Sept. 2017](#a10)* [Week 2-4 in Sep. 2017](#a11)* [Week 1-3 in Oct. 2017](#a12)

Jan. 2017

We came up with the idea about using salt tolerant yeast to dispose kitchen waste which have high osmotic pressure. Then we started to search the papers related to our research.

Mar. 2017

We found three genes that are possible for our goal: *SpTPS1*, *ScTPS1*, and *gltB*.

Apr.2017

And then we began to prepare the materials we need in the following experiments.

May. 2017

We finished the design of nine biobricks with different functions.

Jun. 2017

Designing and editing of the experiment plan.

Transformation of PUC57 plasmid into *DH5α*.

Culture the *E.coli*.

Preparation of plasmid DNA by Alkaline Lysis with SDS;

Digestion (*Xba*Ⅰ, *Spe*Ⅰ).

Recycle the electrophoresis product.

Linearization of the standard carrier.

Week 1-2 in Jul. 2017

Ligate the genes into the standard carrier.

Transform the standard carrier into *DH5α E.coli*;

Culture the *E.coli* and prepare the plasmid.

Week 3 in Jul. 2017

Design the primers of the three genes: *SpTPS1*, *ScTPS1*, and *gltB*.

Week 4 in Jul. 2017 to week 1 in Aug. 2017

PCR PrimeStar DNA polymerase.

Recycle the plasmids from PCR products.

Digest the PCR products.

Electrophoresis and recycling.

Week 2 –week 3 in Aug.2017

Ligation;

We successfully ligated the three target genes in to the vector pPIC9K to get the devices.

Transform the vector into *DH5α E.coli*.

Culture the *E.coli*.

Extract the devices.

Week 4 in Aug. 2017 to week 1 in Sept. 2017

Electrophoresis of six completed devices: intracellular and extracellular expression of the three target genes. Recycle the devices from the gels.

Transform the devices into *GS115* competent yeast cells.

Week 2-4 in Sep. 2017

Sequencing the genes we transformed into the yeast.

Induce experiments by using methanol.

SDS-Page.

Week 1-3 in Oct. 2017

Test the survive rate of the yeast in different salinity.

Test the decomposition rate of the waste.

Sponsors

---

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# Team:CIEI-China/Experiments

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

##### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

##### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:CIEI-China/Results

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* Project
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  + [Achievement](https://2017.igem.org/Team%3ACIEI-China/Achievements)
* Notebook
  + [Lab\_Book](https://2017.igem.org/Team%3ACIEI-China/Notebook)
  + [Protocols](https://2017.igem.org/Team%3ACIEI-China/Protocols)
* [Safety](https://2017.igem.org/Team%3ACIEI-China/Safety)
* Human Practice
  + [Our Story](https://2017.igem.org/Team%3ACIEI-China/HP/Our%20Story)
  + [Silver](https://2017.igem.org/Team%3ACIEI-China/HP/Silver)
  + [Gold](https://2017.igem.org/Team%3ACIEI-China/HP/Gold_Integrated)

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Results

* [*SpTPS1*](#a1)
  + [Optimization](#a2)
  + [Amplification of *ScTPS1*](#a3)
  + [Construction of the device](#a4)
  + [SDS-Page](#a5)
  + [Salinity of the food waste](#a6)
  + [The survivability results](#a7)
* [*SpTPS1*](#a8)
  + [Optimization of *SpTPS1*](#a9)
  + [Amplification](#a10)
  + [Construction of the device](#a11)
  + [Sequence](#a12)
  + [SDS-Page](#a13)
* [*gltB*](#a14)
  + [Optimization](#a15)
  + [Amplification of *gltB*](#a16)
  + [Construction of the device on PIC9K](#a17)
  + [Sequence](#a18)
  + [SDS-Page](#a19)

Results

*SpTPS1*

*Saccharomyces cerevisiae trehalose-6-phosphate synthase* is commonly used by the cells to enhance their tolerance in hypertonic environments. *SpTPS1* is the essential gene in synthesis of trehalose. As a small molecular substance, *SpTPS1* can increase the cell’s ability of regulating the osmotic pressure. Changes in expression of *SpTPS1* can significantly affect the amount of trehalose production in the cell. This gene is originated from the yeast, which have a significant effect on production of trehalose in the yeast.

Optimization

Sequence before Optimization

Saccharomyces cerevisiae S288c chromosome II

ATGACTACGGATAACGCTAAGGCGCAACTGACCTCGTCTTCAGGGGGTAACATTATTGTGGTGTCCAACAGGCTTCCCGTGACAATCACTAAAAACAGCAGTACGGGACAGTACGAGTACGCAATGTCGTCCGGAGGGCTGGTCACGGCGTTGGAAGGGTTGAAGAAGACGTACACTTTCAAGTGGTTCGGATGGCCTGGGCTAGAGATTCCTGACGATGAGAAGGATCAGGTGAGGAAGGACTTGCTGGAAAAGTTTAATGCCGTACCCATCTTCCTGAGCGATGAAATCGCAGACTTACACTACAACGGGTTCAGTAATTCTATTCTATGGCCGTTATTCCATTACCATCCTGGTGAGATCAATTTCGACGAGAATGCGTGGTTGGCATACAACGAGGCAAACCAGACGTTCACCAACGAGATTGCTAAGACTATGAACCATAACGATTTAATCTGGGTGCATGATTACCATTTGATGTTGGTTCCGGAAATGTTGAGAGTCAAGATTCACGAGAAGCAACTGCAAAACGTTAAGGTCGGGTGGTTCTGCACACACCATTCCCTTCGAGTGAAATTTACAGAATCTTACCTGTCAGACAAGAGATTTTGAAGGGTGTTTTGAGTTGTGATTTAGTCGGGTTCCACACATACGATTATGCAAGACATTTCTTGTCTTCCGTGCAAAGAGTGCTTAACGTGAACACATTGCCTAATGGGGTGGAATACCAGGGCAGATTCGTTAACGTAGGGGCCTTCCTATCGGTATCGACGTGGACAAGTTCACCGATGGGTTGAAAAAGGAATCCGTACAAAAGA GAATCCAACAATTGAAGGAAACTTTCAAGGGCTGCAAGATCATAGTTGGTGTCGACAGGCTGGATTACATCAAAGGTGTGCCTCAGAAGTTGCACGCCATGGAAGTGTTTCTGAACGAGCATCCAGAATGGAGGGGCAAGGTTGTTCTGGTACAGGTTGCAGTGCCAAGTCGTGGAGATGTGGAAGAGTACCAATATTTAAGATCTGTGGTCAATGAGTTGGTCGGTAGAATCAACGGTCAGTTCGGTACTGTGGAATTCGTCCCCATCCATTTCATGCACAAGTCTATACCATTTGAAGAGCTGATTTCGTTATATGCTGTGAGCGATGTTTGTTTGGTCTCGTCCACCGTGATGGTATGAACTTGGTTCCTACGAATATATTGCTTGCCAAGAAGAAAAGAAAGGTTCCTTAATCCTGAGTGAGTTCACAGGTGCCGCACAATCCTTGAATGGTGCTATTATTGTAAATCCTTGGAACACCGATGATCTTTCTGATGCCATCAACGAGGCCTTGACTTTGCCCGATGTAAAGAAAGAAGTTAACTGGGAAAAACTTTACAAATACTCTCTAAATA CACTTCTGCCTTCTGGGGTGAAAATTTCGTCCATGAATTATACAGTACATCATCAAGCTCAACAAGCTCCTCTGCCACCAAAAACTGA

Optimization

1. Avoid the restriction enzyme cutting sites *Eco*RⅠ, *Xba*Ⅰ, *Spe*Ⅰ, *Pst*Ⅰ to make sure the following operations on the standard carrier, pSB1C3, can work effectively.

2. Avoid other common restriction enzyme cutting sites: *Bam*HⅠ, *Hin*dⅢ,  *Kpn* Ⅰ,  *Noco* Ⅰ,  *Nde* Ⅰ,  *Not* Ⅰ,  *Sal* Ⅰ,  *Xho* Ⅰ.

3. Add the restriction enzyme cutting sites,  *Xba* Ⅰ and  *Spe* Ⅰ at the beginning and the end of the gene to ligate it with the standard carrier conveniently.

4. We are aimed to enhance the tolerance of  *saccharomyces cerevisiae* ’s in hypertonic environment, so we optimized the sequence to get the highest efficiency by using the  *saccharomyces cerevisiae*  favored codons. In this way, we made sure that the basic sequence will express the same amino acid sequence as in the previous gene before optimization.

5. The *SpTPS1* gene is successfully optimized.

*SpTPS1*

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Sponsors

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