iGEM 2017 — Team RDFZ-China

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# Team:RDFZ-China/Notebook

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MENU

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TEAM

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[Collaborations](https://2017.igem.org/Team%3ARDFZ-China/Collaborations)

PROJECT

[Description](https://2017.igem.org/Team%3ARDFZ-China/Description)
[Design](https://2017.igem.org/Team%3ARDFZ-China/Design)
[Experiments](https://2017.igem.org/Team%3ARDFZ-China/Experiments)
[Notebook](https://2017.igem.org/Team%3ARDFZ-China/Notebook)
[InterLab](https://2017.igem.org/Team%3ARDFZ-China/InterLab)
[Contribution](https://2017.igem.org/Team%3ARDFZ-China/Contribution)
[Model](https://2017.igem.org/Team%3ARDFZ-China/Model)
[Results](https://2017.igem.org/Team%3ARDFZ-China/Results)
[Demonstrate](https://2017.igem.org/Team%3ARDFZ-China/Demonstrate)
[Improve](https://2017.igem.org/Team%3ARDFZ-China/Improve)
[Attributions](https://2017.igem.org/Team%3ARDFZ-China/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3ARDFZ-China/Parts)
[Basic Parts](https://2017.igem.org/Team%3ARDFZ-China/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3ARDFZ-China/Composite_Part)
[Part Collection](https://2017.igem.org/Team%3ARDFZ-China/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3ARDFZ-China/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3ARDFZ-China/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3ARDFZ-China/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3ARDFZ-China/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3ARDFZ-China/Applied_Design)
[Entrepreneurship](https://2017.igem.org/Team%3ARDFZ-China/Entrepreneurship)
[Hardware](https://2017.igem.org/Team%3ARDFZ-China/Hardware)
[Measurement](https://2017.igem.org/Team%3ARDFZ-China/Measurement)
[Model](https://2017.igem.org/Team%3ARDFZ-China/Model)
[Plant](https://2017.igem.org/Team%3ARDFZ-China/Plant)
[Software](https://2017.igem.org/Team%3ARDFZ-China/Software)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=RDFZ-China)

# RDFZ-China

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* [Team](https://2017.igem.org/Team%3ARDFZ-China/Team)
  + [Results](https://2017.igem.org/Team%3ARDFZ-China/Results)
  + [Collobration](https://2017.igem.org/Team%3ARDFZ-China/Collaboration)
  + [Interlab](https://2017.igem.org/Team%3ARDFZ-China/InterLab)
* [Project](https://2017.igem.org/Team%3ARDFZ-China/Description)
  + [Description](https://2017.igem.org/Team%3ARDFZ-China/Description)
  + [Design](https://2017.igem.org/Team%3ARDFZ-China/Design)
  + [Experiments](https://2017.igem.org/Team%3ARDFZ-China/Experiments)
  + [Parts](https://2017.igem.org/Team%3ARDFZ-China/Parts)
* [Model](https://2017.igem.org/Team%3ARDFZ-China/Model)
* [Safety](https://2017.igem.org/Team%3ARDFZ-China/Safety)
* [Human Practice](https://2017.igem.org/Team%3ARDFZ-China/HP/Silver)
  + [Notebook](https://2017.igem.org/Team%3ARDFZ-China/Notebook)
* [Awards](https://2017.igem.org/Team%3ARDFZ-China/Applied_Design)
  + [Model](https://2017.igem.org/Team%3ARDFZ-China/Model)
  + [Entrepreneurship](https://2017.igem.org/Team%3ARDFZ-China/Entrepreneurship)

7.23
1、PCR for BioBricks, 5’ homologous arm, backbone
2、electrophoresis：Validate PCR product
3、枯草杆菌保种
7.24
1、Get genome
2、昨日PCR产物回收
3、Genome PCR
5、引物退火
6、transcribe, replicate pVeg
7.26
1、electrophoresis（genome，退火产物）
8.3
1、enzyme digestion: pHT01, using BamHI & XbaI
2、electrophoresis: testing the product of enzyme digestion（PHT01，PHT01 enzyme digestion product，pVeg PCR, pVeg PCR enzyme digestion product）
10、胶回收
8.4
1、PCR（pVeg，GFP, negative control，positive control）
2、 electrophoresis: test the product of PCR
5、enzyme digestion: YerP, gfp, sfp, pSB1C3
6、Ligation
7、PCR产物回收
8、transcription
8.5
1、enzyme digestion: pVeg, RBS, Backbone
2、Colony PCR（sfp2 A 抗, YerP1 A抗, YerP2 A抗）
8.9
1、electrophoresis: 菌PCR
2、get genome，ER，尝试双酶切验证质粒
8.10
1、B.sub 感受态制备
2、8月9号转化涂板因未吹干失败，菌落被感染
8.14
1、enzyme digestion: sfp，pSB1C3
2、electrophoresis: test the product of胶纯化
3、transcribe RFP Construct（control）
4、ligate 19-TC 载体，product（YerP /sfp），Solution Ⅰ Control
8.21
1、PCR（sfp，YerP，pVeg，positive control，negative control）
3、ligation pVeg-RBS A抗
8.21
1、Transformation（pVeg DH5α C抗，pVeg RBF DH5α A抗，D6B HP K抗，PUC19 对照A抗（稀），rfp枯草未知1，rfp枯草未知2，PUC19对照A抗（浓）, pVeg DH5α A抗，rfp DH5α K抗，pVeg RBS A抗，sfp, YerP）
8.22
1、Taq Super-mix
2、electrophoresis
3、Digestion
4、Ligation
8.23
1、electrophoresis（D2000，1kb Plus Ladder，pSB1A3-GFP，pSB1C3-pTet-GFP，positive control，negative control）
2、digestion for validation
3、electrophoresis（ladder， pveg，pveg切，pveg-RBS，pveg-RBS 切，pH101-RFP，pH101-RFP切，T-sfp，T-sfp 旧，T-YerP，sfp-RBS）
9.2
1、PCR plasmid（T-sfp提，T-YerP提，sfp-RBS提，pvg-RBS提，T-sfp连，T-YerP连，sfp-RBS连，pvg-RBS连，pveg-RBS）
2、electrophoresis（ladder，pSB1A3-GFPpSB1C3-pTet-GFP丙sfp2 A 抗, 庚， YerP1 A抗, 辛, negative control，positive control）（ladder，TSFP1，TSFP2，TSFP3，TSFP4，TSFP5）
（ladder，T-SFP，T-SFP切，sfp-RBS，sfp-RBS切，pVg-RBS，pVg-RBS切，T-YerP，T-YerP切，PHT01-GFP，PHT01-GFP切）（ladder，pSB1A3-GFP，己，T-YerP1，T-YerP2，T-YerP3，T-YerP4，T-YerP5，T-YerP6，T-YerP7）
第19页左半部分不知道说什么
第20页最上面有个5号可能是电泳
9.4
1、transcribe T-sfp，T-YerP
9.5
1、PCR（5‘Homologous arm，3’Homologous arm，backbone，BioBricks site）
2、胶回收
3、Transcribe（T-sfp，Y-YerP，pVeg，positive control）
9.8
1、PCR
2、PCR:T-sfp
3、enzyme digestion（E、P），（PSB1C3-rfp plasmid）
4、胶回收
5、验证（ladder，5‘homologous arm，3‘homologous arm，bb，RFP construct）
9.9
1、electrophoresis: test pSB1C3-rfp回收plasmid及7.26PCR得YerP
2、Pfu扩增Biobrick sites及k位点
3、上样（1-6 6管PSB1C3-rfp, 7-8 7.26 YerP，PSB1A3-gfp质粒）
4、YerP平板取菌划线，菌落PCR
7、get plasmid→PSB1C3-rfp construct，PCR
9、electrophoresis: test product of PSB1C3-rfp 回收产物
10、凝胶回收（sfp-biobrick, pVeg）
9.9
1、胶回收
2、electrophoresis1（D2000, RFP1,RFP2,RFP3,Bsub2,Bsub3,Bsub4,Busb5,Bsub6）
3、electrophoresis（D2000, Biobrick，sfp，Pveg）
9.12
1、enzyme digestion（PSB1C3-rfp）
2、electrophoresis——胶回收
（左：ladder，bs，5‘homologous arm，3‘homologous arm，BioBricks site，pSB1C3）
（右：ladder，YerP1，YerP2，YerP3，YerP4，pSB1C3）
3、test（ladder，pVeg-RBS，sfp-RBS，sfp-bb，A3，A4，A5，A6）
9.13
1、胶回收
2、PCR（pVeg-RBS，sfp-RBS，ext2）
3、7管PHT01酶切检测
4、第二轮RBS PCR体系
6、electrophoresis：胶回收产物回收（ladder，YerP1，YerP2，YerP3，YerP4，BioBricks site，Backbone，5‘ homologous arm，3‘ homologous arm，BioBricks site）
PCR产物
1、 ladder-D2000
2、 pVg-RBS-ext 1
3、 pVg-RBS-ext 2
4、 sfp-RBS-ext 1
5、 sfp-RBS-ext 2
6、 PSB1C3回收
9.14
1、electrophoresis （ladder：1kb plus DNA）
2、pVeg-RBS-ext2
3、sfp-RBS-ext2
4、pHT01 1
5、pHT01 2
9.15
1、pfu: erP+BioBricks site PCR
2、加A
3、YerP 加A
4、electrophoresis（1kb ladder，YerP1 bio，YerP2 bio）
5、electrophoresis（ladder 1kb plus，pVeg-RBS 加A 回收，sfp-RBS 加A 回收，YerP1 回收，YerP2 回收，PSB1C-sfp，RFP3）
6、连接
9.17
1、transcribe: Bsub
2、transcribe: T-YerP
3、OEPCR1
9.18
1、枯草感受态检测——菌落数量
2、回收pVeg+sfp OEPCR
3、重叠PCR体系
9.20
1、OEPCR3
2、菌落PCR（psb1C3-AH）
3、菌PCR electrophoresis（ladder，YerP，sfp，GFP，negative control，positive control，6）
4、回收1000kb左右的pvg-sfp
5、回收OEPCR3
9.21
1、新backbone，BioBricks site用pfu和2017○34GP3
9.22
1、YerP PCR
2、胶回收
9.25
1、PCR：backbone，BioBricks site
9.26
1、OEPCR3 加A
2、electrophoresis（none，ladder D2000，BioBricks site，backbone）
9.27
1、electrophoresis: for testing the product of回收产物
First: 2-5道：T-YerP○5 1-4； 6-9道：T-YerP○4 1-4）
Second:（2-5道：AH，1-4；6-7道：BioBricks site，Backbone）
2、transcribe pVeg-bfp
9.28
1、菌PCR（ladder D2000，T-YerP 10，T-YerP 11，T-YerP 9，T-YerP 5，T-YerP 4，T-YerP pvg-sfp，T-YerP粉，划线5，划线4）
9.30
1、菌液PCR
2、Gibson片段-pveg-sfp PCR）
3、electrophoresis（1，2，3，4，5，pveg sfp gibs，+，-）
10.9
1、回收Gib-（pveg-sfp）
2、enzyme digestion pVeg-sfp
3、ligate pSB1C3-pvg-sfp
10.10
1、electrophoresis（ladder，YerP1，YerP2，YerP3，YerP4，5‘ homologous arm，3‘ homologous arm，Gb，bb，BB）
10.11
1、electrophoresis（D2000，1，2，3，4，5，6，7，8，9，10）
2、electrophoresis（D2000，11，12，13，14，15，16，17，18）
10.16
1、electrophoresis（ladder D2000，OEPCR3）
2、transcribe（control，T-pveg sfp rbs，IDT’s ImrA）
3、electrophoresis testing（1kb plus，OEPCR 回收产物，5‘，3‘，pSB1C3切, p SB1C3）
10.18
1、PCR菌（○1pSB1A3-GFPpSB1C3-pTet-GFP丙丁戊阴○2psb-pvg-sfp连接产物，OEPCR，RFP3，P3FG）
10.19
1、菌PCR（○1ladder 2000，sfp，GFP，negative control，positive control，6）（D2000，psb-pvg-sfp连接产物，OEPCR，RFP3，P3FG）
10.21
1、PCR
2、enzyme digestion（psb1C3，RFP3，lmrA质粒，pvg-sfp）
3、electrophoresis（ladder 1kb plus，OEPCR3，ImrA1，ImrA2，D1，E1）
4、electrophoresis（ladder 1kb plus，A1，B1，C1）
10.22
1、37度恒温箱里（lmrA切1，2（E，P得PCR产物），超薄试剂盒纯化，验证
2、Ligate PSB1C3-lmrA
3、 Transcribe PSB1C3-pveg-sfp转化得平板
4、Gibson
6、gov electrophoresis（1kb plus marker，lmrA切1，2回收，lmrA2\*2，lmrA3\*2，pvg sfp\*2，PSB1C3线性，pSB1C3）
3、菌PCR（D2000，浓pveg-sfp，OEPCR，阳）
4、菌PCR（D2000，YerP，sfp）
10.23
1、electrophoresis（D2000, ImrA2，lmrA2，lmrA3，lmrA4）
10.24
1、electrophoresis（ladder 1kb plus，lmrA2，lmrA3，lmrA4，1-1 pvg-rbs，2-1 pvg-rbs，2-3 pvg-rbs，1-2 pvg-rbs，1-3 pvg-rbs，RFP

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# Team:RDFZ-China/Experiments

RDFZ-China

![](https://static.igem.org/mediawiki/2017/5/57/RDFZ_logo.png)

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* [Team](https://2017.igem.org/Team%3ARDFZ-China/Team)
  + [Attribution](https://2017.igem.org/Team%3ARDFZ-China/Attributions)
  + [Results](https://2017.igem.org/Team%3ARDFZ-China/Results)
  + [Safety](https://2017.igem.org/Team%3ARDFZ-China/Safety)
  + [Interlab](https://2017.igem.org/Team%3ARDFZ-China/InterLab)
  + [Notebook](https://2017.igem.org/Team%3ARDFZ-China/Notebook)
* [Project](https://2017.igem.org/Team%3ARDFZ-China/Description)
  + [Description](https://2017.igem.org/Team%3ARDFZ-China/Description)
  + [Design](https://2017.igem.org/Team%3ARDFZ-China/Design)
  + [Experiments](https://2017.igem.org/Team%3ARDFZ-China/Experiments)
* [Awards](https://2017.igem.org/Team%3ARDFZ-China/Applied_Design)
  + [Entrepreneurship](https://2017.igem.org/Team%3ARDFZ-China/Entrepreneurship)
* [Parts](https://2017.igem.org/Team%3ARDFZ-China/Parts)
  + [Basic Parts](https://2017.igem.org/Team%3ARDFZ-China/Basic_Part)
  + [Composite Parts](https://2017.igem.org/Team%3ARDFZ-China/Composite_Part)
* [Human
  Practice](https://2017.igem.org/Team%3ARDFZ-China/HP/Silver)
  + [HP Silver](https://2017.igem.org/Team%3ARDFZ-China/HP/Silver)
  + [HP Gold](https://2017.igem.org/Team%3ARDFZ-China/HP/Gold_Integrated)
  + [Public Engagament](https://2017.igem.org/Team%3ARDFZ-China/Engagement)
  + [Collobration](https://2017.igem.org/Team%3ARDFZ-China/Collaboration)
* [Model](https://2017.igem.org/Team%3ARDFZ-China/Model)
  + [Hardware](https://2017.igem.org/Team%3ARDFZ-China/Hardware)

![](https://static.igem.org/mediawiki/2017/6/63/Rdfz_wiki_experiment.png)

1 / 3

![](https://static.igem.org/mediawiki/2017/5/5c/Rdfz_experiment_in_lab.jpeg)

2 / 3

![](https://static.igem.org/mediawiki/2017/d/d4/Rdfz_interlab_study_picture.jpeg)

3 / 3

![](https://static.igem.org/mediawiki/2017/7/7a/RdFZ-China_Lab_1.jpeg)

❮
❯

# Assembly

---

We hereby describe the assembly process involved in our project:

![](https://static.igem.org/mediawiki/2017/6/66/2017RDFZ_Assembly.png)

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# Team:RDFZ-China/Results

RDFZ-China

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![](https://static.igem.org/mediawiki/2017/6/6e/RDFZ_wiki_nav.png)

* [Team](https://2017.igem.org/Team%3ARDFZ-China/Team)
  + [Attribution](https://2017.igem.org/Team%3ARDFZ-China/Attributions)
  + [Results](https://2017.igem.org/Team%3ARDFZ-China/Results)
  + [Safety](https://2017.igem.org/Team%3ARDFZ-China/Safety)
  + [Interlab](https://2017.igem.org/Team%3ARDFZ-China/InterLab)
  + [Notebook](https://2017.igem.org/Team%3ARDFZ-China/Notebook)
* [Project](https://2017.igem.org/Team%3ARDFZ-China/Description)
  + [Description](https://2017.igem.org/Team%3ARDFZ-China/Description)
  + [Design](https://2017.igem.org/Team%3ARDFZ-China/Design)
  + [Experiments](https://2017.igem.org/Team%3ARDFZ-China/Experiments)
* [Awards](https://2017.igem.org/Team%3ARDFZ-China/Applied_Design)
  + [Entrepreneurship](https://2017.igem.org/Team%3ARDFZ-China/Entrepreneurship)
* [Parts](https://2017.igem.org/Team%3ARDFZ-China/Parts)
  + [Basic Parts](https://2017.igem.org/Team%3ARDFZ-China/Basic_Part)
  + [Composite Parts](https://2017.igem.org/Team%3ARDFZ-China/Composite_Part)
* [Human
  Practice](https://2017.igem.org/Team%3ARDFZ-China/HP/Silver)
  + [HP Silver](https://2017.igem.org/Team%3ARDFZ-China/HP/Silver)
  + [HP Gold](https://2017.igem.org/Team%3ARDFZ-China/HP/Gold_Integrated)
  + [Public Engagament](https://2017.igem.org/Team%3ARDFZ-China/Engagement)
  + [Collobration](https://2017.igem.org/Team%3ARDFZ-China/Collaboration)
* [Model](https://2017.igem.org/Team%3ARDFZ-China/Model)
  + [Hardware](https://2017.igem.org/Team%3ARDFZ-China/Hardware)

![](https://static.igem.org/mediawiki/2017/c/c0/Rdfz_wiki_results.png)

# Achievements

---

We have successful results:

* We helped to generate the data for the Interlab study this year.
* We isolated two full-length genes - sfp and YerP from our Bacillus subtilis strain and successfully processed them into biobricks, and the sequencing results are positive.
* We ligated lmrA into pHT01, a dual host, expression vector for Bacillus subtilis, and the sequencing result is positive.
  We measured the growth curve for our strain and tested two different transformation protocols.
* We assembled our surfactin-producing operon by overlap extension PCR, and the sequencing results are positive.
* We constructed a mathematical model and applied various estimations and confirmed parameters to obtain useful predictions.
* We designed and constructed a sprinkler model that can be used to spray bacterial suspensions for bioremediation.
* We adapted a protocol to measure leaching efficiency of different eluents in the soil quantitatively.

But we also have unsuccessful results:

* We failed to ligate many of our biobricks into pSB1C3 at various times, which may be due to low ligation efficiency in our lab.
* We didn’t do much characterization for our parts.
* We didn’t have time to test for surfactin production by our engineered strain.
* We didn’t construct our designed integration vector for B. subtilis, although we had prepared the fragments for Gibson assembly.
* We had a difficult time of transforming B. subtilis in our lab, and we once contaminated the competent cells.

# Future Plans

---

* We will complete the construction of our integration vector and surfactin-producing operon.
* We will test for the efficiency of surfactin production by analytical methods.
* We will refine our protocol for testing leaching efficiency and test our strain’s ability to emulsify the oil.
* We will analyze the eluate to see if the microbiome degrades the hydrocarbons.
* We will deposit our strain in a real-world contamination zone and see if it works.
* We will continue to analyze possible interactions between lmrA and surfactin as its substrate; then we will find mutated variants that could export surfactin more efficiently.

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