iGEM 2015 — Team NEFU_China

# Team:NEFU China/Notebook

**Monthly**

---

**January**

Our junior and senior students were attracted by the recruiting announcement for the iGEM 2015 in our university. After several interviews and competitive selections, our team was basically created.

**February**

Our team was growing. We communicated with previous iGEMers in our school to get more insights into iGEM. Meanwhile, we started to watch successful iGEM cases of previous years.

**March**

We wandered among wikis and gradually realized what the basic factors are for a good project. Brainstorms and weekly meet-ups promoted us to discover the quintessence of iGEM.

**April**

We urged ourselves to get deeper insights into those previous cases to catch their inspiration. After meet-ups with teammates and our instructors, a draft idea of a yogurt guarder came to our mind. To make sure our project was worth proceeding, we did a survey to find out how much attention people would pay to the quality of yogurt. Fortunately, we got the expected results, although we knew we still had a long way to go.

**May**

We primarily designed the basic structure of our project. Then we read numerous papers to make an integrated protocol of our lab work.Besides, we visited a dairy factory to learn the microbiological detection methods used in practical production of yogurt. We discovered that our method to be developed should have advantages over the traditional methods, especially in portability.
In addition, it was essential for us to adapt to the wet lab work. We started with laboratory safety training. With the instruction of our instructors and previous iGEMers, we learned a lot of techniques in molecular biology.

**June**

With efforts, our wet lab work went smoothly.

**July**

This month, we organized public activity to popularize the knowledge of yogurt production and its quality inspection, and remind people to store yogurt properly.
In addition, we did a survey to confirm the key factors which will attract customers’ attention if our yogurt guarder is developed into a real product.

**August**

On 13th-15th, we attended the summit of Central China iGEM Consortium held by Peking University. We shared our experience with other teams and really learned a lot from other teams. Here we want to express our sincere thanks to them!
Meanwhile, our lab works still proceeded. We encountered difficulty in the electrotransformation of bacteria, which delayed our project but was eventually resolved after multiple attempts.

**September**

Time flies. We seized the time to make everything done and put great efforts to the wiki building and preparation for the Giant Jamboree.

**Labnote**

---

Isolation of genomic DNA from bacteria             The genomic DNA of *Salmonella enterica* subsp. *enterica serovar Typhimurium* str. LT2 and*Lactobacillus delbrueckii* subsp.*Bulgaricus* str. Lb14 were isolated using the TIANamp Bacteria DNA Kit (see protocol). The concentration of genomic DNA was measured with a NanoDrop spectrophotometer.

Strain identification

We applied 16S ribosomal DNA identification to identify the strain of Salmonella and Lactobacillus bought from a biotech company. Universal primers were designed according to the conserved sequences.

A. Identification of Salmonella typhimurium

1.PCR

![](https://static.igem.org/mediawiki/2015/0/05/NEFU_China_5351A137-7EAB-4153-B27D-FDEDBACC8656.png)

![](https://static.igem.org/mediawiki/2015/0/08/NEFU_China_1A00253D-C888-4317-83AB-12FBD6D8F14E.png)

                Fig 1. 1. Marker (2000bp DNA Ladder) ; 2. *Salmonella typhimurium* 16s rRNA

2. Ligation (TaKaRa DNA Ligation Kit see protocol)

![](https://static.igem.org/mediawiki/2015/6/6c/NEFU_China_CD600935-F7B2-41DF-AE7A-1356B2D74459.png)

3. Transformation ([see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf))

4. Colony PCR

![](https://static.igem.org/mediawiki/2015/f/fa/NEFU_China_80678D69-C533-4A86-A51D-001697EC74D8.png)

                                     ![](https://static.igem.org/mediawiki/2015/c/ce/NEFU_China_418396AA-6D58-4C22-ADA9-5820CEC9AD9A.png)

Fig 2. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony; 6. Negative control

5.Sequencing（Show results）

6.We then did alignment using BLAST.

![](https://static.igem.org/mediawiki/2015/a/af/NEFU_China_E5ADCBCB-A89F-436F-AEBC-929E7CD914BD.png)

 The sequence was consist with that of *Salmonella enterica* subsp. *enterica serovar Typhimurium* str. LT2.

B. Identification of Lactobacillus bulgaricus

1. PCR

![](https://static.igem.org/mediawiki/2015/6/6d/NEFU_China_D7858136-01CC-46B3-81F6-968BA3780EF0.png)

![](https://static.igem.org/mediawiki/2015/b/b5/NEFU_China_3722D6C0-A70A-4DBB-B538-ED40DC3D889D.png)

Fig 3. 1. Marker (DL2000) ; 2. *Lactobacillus bulgaricus*16s rRNA

2. Ligation (TaKaRa DNA Ligation Kit see protocol)

![](https://static.igem.org/mediawiki/2015/d/d6/NEFU_China_1C19B503-1B40-4A7E-A648-1BCD39B79DAC.png)

3. Transformation ([see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf))

4. Colony PCR

![](https://static.igem.org/mediawiki/2015/1/10/NEFU_China_A09473EB-D1C4-4E0E-84C1-166D737F2C94.png)

![](https://static.igem.org/mediawiki/2015/6/6a/NEFU_China_087DB8ED-A25B-471E-903F-30722BDBA545.png)

Fig 4. 1. Marker (DL2000) ; 2.3.4.5.6 each for one single colony; 7. Negative control

5. Sequencing（Show results）

6. We then did alignment using BLAST.

The sequence was almost consist with that of *Lactobacillus delbrueckii* subsp.*Bulgaricus* str. Lb14. The differences were caused by subculturing perhaps.

Gene Cloning of*Lsr A, B, C, D, R*and *K*

1. Gene isolation using PCR

(Restriction enzyme sites are indicated in red font)

*lsrA*

![](https://static.igem.org/mediawiki/2015/3/3c/NEFU_China_890BDFC2-9990-40D4-897D-CFA4E7E07181.png)

*lsrB*

![](https://static.igem.org/mediawiki/2015/2/23/NEFU_China_E854482B-4DC9-40E3-8445-9FF5E79E7764.png)

*lsrC*

![](https://static.igem.org/mediawiki/2015/1/1c/NEFU_China_084987BB-1EBE-415F-9049-FEFEA75EC092.png)

*lsrD*

![](https://static.igem.org/mediawiki/2015/6/66/NEFU_China_B6FF9108-6231-4FE1-9032-6A754A410CC8.png)

*lsrK*

![](https://static.igem.org/mediawiki/2015/0/0b/NEFU_China_FD688B56-317E-43BD-8B4B-39766082AAB0.png)

*lsrR*

![](https://static.igem.org/mediawiki/2015/f/f4/NEFU_China_EE311814-A6B0-4227-ACFC-90A1272E3097.png)

![](https://static.igem.org/mediawiki/2015/1/1c/NEFU_China_9D7F3095-FE86-4E6D-853C-681D7A4B603F.jpg)

Fig 5. PCR products of lsrA, B and C  1.Marker (DL2000) ; 2.lsrA (1536bp) ; 3.Negative control;

4.lsrB (1023bp) ; 5.Negative control; 6.lsrC (1089bp) ; 7.Negative control

                                                                                                             ![](https://static.igem.org/mediawiki/2015/d/dd/NEFU_China_0954A0AA-5C06-49C4-A6EE-F63B36DE7E69.jpg)

Fig 6. PCR products of lsrD, R and K  1.Marker (DL2000) ; 2.lsrD (1002bp) ; 3.Negative control;

4.lsrR (960bp) ; 5.Negative control; 6.lsrK (1593bp) ; 7.Negative control

2.Ligation (TaKaRa DNA Ligation Kit see protocol)

![](https://static.igem.org/mediawiki/2015/a/a3/NEFU_China_B2E2091E-C260-4EE2-949B-3EA94A5E2D1D.png)![](https://static.igem.org/mediawiki/2015/a/a3/NEFU_China_B2E2091E-C260-4EE2-949B-3EA94A5E2D1D.png)

3.Transformation ([see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf)）

4.Colony PCR

*lsrA*

![](https://static.igem.org/mediawiki/2015/d/d8/NEFU_China_9C5A87D7-9823-4A7F-A364-2BF5A0F9577B.png)

![](https://static.igem.org/mediawiki/2015/b/ba/NEFU_China_B8CBC470-E785-4DF8-8F2E-DE83D315F6EB.jpg)

Fig 7. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony (1536bp) ; 6.Negative control

*lsrB*

![](https://static.igem.org/mediawiki/2015/3/3f/NEFU_China_D69BDDE6-652A-41F4-8DDA-7ACC82AAF452.png)

![](https://static.igem.org/mediawiki/2015/5/50/NEFU_China_5818DF91-9376-471B-8B85-7967C5F0F3B7.jpg)

Fig 8. Clony PCR results. 1. Marker (DL2000) ; 2.3.4 each for one single colony (1023) ; 5.Negative control

*lsrC*

**![](https://static.igem.org/mediawiki/2015/6/6f/NEFU_China_BAC45DDC-FF3C-4A95-9076-4080D545E24D.png)**

![](https://static.igem.org/mediawiki/2015/5/5e/NEFU_China_406D2C32-9C2C-479E-A080-414583438808.jpg)

Fig 9. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony (1089bp) ; 6.Negative control

*lsrD*

![](https://static.igem.org/mediawiki/2015/e/eb/NEFU_China_3B850D6B-F363-4603-95DC-E802D5BB387B.png)

![](https://static.igem.org/mediawiki/2015/8/86/NEFU_China_705C5ED8-2845-48E8-B4F2-574B24E8CD6B.jpg)

Fig 10. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony (1002bp) ; 6.Negative control

*lsrK*

![](https://static.igem.org/mediawiki/2015/7/77/NEFU_China_B80CF3CA-0BE7-4DAB-87D7-B77B87B361BA.png)

![](https://static.igem.org/mediawiki/2015/5/59/NEFU_China_E346D693-FE67-4890-A9AB-86FAD9F8068D.jpg)

Fig 11. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony(1593bp) ; 6.Negative control

*lsrR*

![](https://static.igem.org/mediawiki/2015/c/ca/NEFU_China_4EE6A139-BDFC-4DBB-AF24-2C55019DDE48.png)

![](https://static.igem.org/mediawiki/2015/8/83/NEFU_China_0E266A05-C035-4B64-A7F3-2A86DD198974.jpg)

Fig 12. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony; 6.Negative control

5.sequencing

![](https://static.igem.org/mediawiki/2015/8/85/NEFU_China_1D4FCA4D-2694-4D92-BAE4-D29C27A89915.png)

Fig 13. Sequencing result of LsrA

![](https://static.igem.org/mediawiki/2015/c/c5/NEFU_China_6BD935D8-F97F-4490-81E5-6A5ECBB665FC.png)

Fig 14. Sequencing result of LsrB

![](https://static.igem.org/mediawiki/2015/2/21/NEFU_China_82ED9097-982D-454B-98B5-AB52A5211EF1.png)

Fig 15. Sequencing result of LsrC

![](https://static.igem.org/mediawiki/2015/d/df/NEFU_China_3C6BEB94-FDC3-410E-AEE9-676CD8312D95.png)

Fig 16. Sequencing result of LsrD

![](https://static.igem.org/mediawiki/2015/8/80/NEFU_China_19EA3109-A08F-4BBD-8D6A-723289063F0E.png)

Fig 17. Sequencing result of LsrK

![](https://static.igem.org/mediawiki/2015/4/4f/NEFU_China_B61B8629-374E-4FC6-AF63-A2E44E7BA32F.png)

Fig 18. Sequencing result of LsrR

All the sequencing results were consistent with our designation, except for LsrD. There was a wrong base pair in the sequences we cloned, so we chose SOE(Splicing by overlap extension)-PCR to fix this error.

A. Primary PCR reaction

Segment1-lsrD1

![](https://static.igem.org/mediawiki/2015/d/da/NEFU_China_8B1F2BCB-3FB8-40F7-98C8-9173416417A8.png)

Segment2-lsrD2

![](https://static.igem.org/mediawiki/2015/0/08/NEFU_China_29EAEE3C-2E1A-41D5-BBA3-7293C61B8124.png)

                                                                                                                                          ![](https://static.igem.org/mediawiki/2015/4/4c/NEFU_China_F957A08D-C3B5-4A21-9DD5-8B1EAD5D8978.png)

Fig 19. 1. Marker (DL10000) ; 1. LsrD1 ; 2. LsrD2

B. Overlapping and elongation

![](https://static.igem.org/mediawiki/2015/3/35/NEFU_China_291C996B-3652-4073-8F85-4351A3B8BCA0.png)

C. Second PCR reaction

![](https://static.igem.org/mediawiki/2015/c/c0/NEFU_China_032E37C2-7F01-4D46-8AE5-18B072CF5100.png)

![](https://static.igem.org/mediawiki/2015/f/fe/NEFU_China_25AE26FA-6322-4D10-B4D3-CFD4EDF349AF.png)

Fig 20. 1. Marker (DL10000) ; 1. LsrD ; 2. LsrD1; 3. LsrD2

Ligation

![](https://static.igem.org/mediawiki/2015/b/bb/NEFU_China_8EF057C0-7F20-44BD-9362-D3CCBBF166BB.png)

Transformation (see protocol)

Clone **pcr**

![](https://static.igem.org/mediawiki/2015/3/3b/NEFU_China_EB92ADF4-1C00-4F5F-BA7F-C0A4111A95C6.png)

![](https://static.igem.org/mediawiki/2015/2/23/NEFU_China_ECFDFF16-BEA0-4F4C-8EF0-D80F6D7942D4.png)

Fig 21. 1. Marker (DL2000) ; 2.3.4.5.each for one single colony(1002bp) ; 6.Negative control

Sequencing

![](https://static.igem.org/mediawiki/2015/9/99/NEFU_China_E6C39C29-FC7F-43D2-ACD5-B1EC15673CD5.png)

As we see, the sequencing result is consistent with our designation.

**Plasmid construction**

 All designed fragments needed in plasmid construction were replicated using PCR .

**1. pNZ8148-lsrA**

**Insert lsrA**

Miniprep (pEASY-T5 cloning vector with LsrA) with TIANprep Mini Plasmid Kit（see protocol）

1.PCR

![](https://static.igem.org/mediawiki/2015/6/6d/NEFU_China_94713099-D0AB-4D58-8DA1-3E62FFA0D8B3.png)

Double digestion (Takara)

![](https://static.igem.org/mediawiki/2015/1/1b/NEFU_China_7A977166-D8B0-420B-9844-F0A58C8CB665.png)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1[see manual](http://www.takara.com.cn/?action=Page&Plat=pdetail&newsid=101&subclass=1))

![](https://static.igem.org/mediawiki/2015/1/17/NEFU_China_39C0A08F-096B-48C1-9764-E5F5816EC6F4.png)

Transformation [see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf)

Colony PCR

![](https://static.igem.org/mediawiki/2015/2/20/NEFU_China_18B85C22-8EF6-4DD2-9E14-40D95C2ABE92.png)

![](https://static.igem.org/mediawiki/2015/e/e7/NEFU_China_0ECC36ED-F12A-4C7E-A93B-FDF1212BEED6.png)

    Fig 22.1. Marker (DL2000) ; 2. Postive control (1536bp) ; 3.4. each for one single colony; 5. Negative control

Two-step enzyme digestion(Takara) for detection

![](https://static.igem.org/mediawiki/2015/a/a3/NEFU_China_B6CBC1C6-A178-4477-99AC-F9AC10532BCD.png)

                                                                                                                                   ![](https://static.igem.org/mediawiki/2015/1/1f/NEFU_China_D8A4081C-855E-4C56-BD09-F7D0AA3900F3.png)

                  Fig 23. 1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1536bp) ;

3. digest vector (3167bp) ; 4.pcr product (1536bp)

The sequencing result is consistent with our designation.

![](https://static.igem.org/mediawiki/2015/b/bc/NEFU_China_0C4F944B-4A12-452D-802E-79563F1A6881.png)

**2. pNZ8148-lsrB**

Miniprep (pEASY-T5 cloning vector with LsrB) with TIANprep Mini Plasmid Kit（see protocol）

**Insert lsrB**

1.PCR

![](https://static.igem.org/mediawiki/2015/2/22/NEFU_China_24BACE72-9696-4E8F-996A-A39F77BCD376.png)

Double digestion (Takara)

![](https://static.igem.org/mediawiki/2015/f/fb/NEFU_China_CDF8CF67-9BA9-4096-BF87-D80C0AE9C68A.png)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1[see manual](http://www.takara.com.cn/?action=Page&Plat=pdetail&newsid=101&subclass=1))

![](https://static.igem.org/mediawiki/2015/1/15/NEFU_China_BA1439C7-3254-4BE4-952D-BE481A8CD057.png)

Transformation [see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf)

Colony PCR

![](https://static.igem.org/mediawiki/2015/d/d8/NEFU_China_3434BA7A-00BA-42E4-B1E8-1A13C4ACF4EA.png)

![](https://static.igem.org/mediawiki/2015/5/5c/NEFU_China_F40B0503-6325-44AF-8C6B-F191A326DCA3.png)

   Fig 24. 1. Marker (DL2000) ; 2. Postive control (1023bp) ; 3.4.5.6.7. each for one single colony; 8. Negative control

Two-step enzyme digestion(Takara) for detection

![](https://static.igem.org/mediawiki/2015/d/df/NEFU_China_2C907F60-37C0-4A41-B0E1-EAE9AF7B66AB.png)

![](https://static.igem.org/mediawiki/2015/1/1f/NEFU_China_48721673-9D75-4F41-84D3-5C6E19FC3E5C.png)

Fig 25. 1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1023bp) ;

3. digest vector (3167bp) ; 4.pcr product (1023bp)

The sequencing result is consistent with our designation.

![](https://static.igem.org/mediawiki/2015/f/ff/NEFU_China_1B150880-5BE0-4E47-91E8-21AF8F84D017.png)

**3. pNZ8148-lsrC**

Miniprep (pEASY-T5 cloning vector with LsrC) with TIANprep Mini Plasmid Kit（ see protocol）

**Insert lsrC**

1.PCR

![](https://static.igem.org/mediawiki/2015/1/1a/NEFU_China_E23AD187-ABD7-4476-A15D-1CE8F6D341C3.png)

Double digestion (Takara)

![](https://static.igem.org/mediawiki/2015/5/5c/NEFU_China_FBA645D3-053B-45F5-BF6C-FB7D9CA02BA4.png)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1[see manual](http://www.takara.com.cn/?action=Page&Plat=pdetail&newsid=101&subclass=1))

![](https://static.igem.org/mediawiki/2015/7/7e/NEFU_China_EF7A8BD4-526E-4523-9CCB-3A647ADB2CAB.png)

Transformation [see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf)

Colony PCR

![](https://static.igem.org/mediawiki/2015/8/83/NEFU_China_ED5282F5-23BE-44D1-AB08-BE640545F7D2.png)

  ![](https://static.igem.org/mediawiki/2015/7/76/NEFU_China_12A38364-2AD5-4C80-98D8-6461FDE0218A.png)

Fig 26. 1. Marker (DL2000) ; 2. Postive control (1044bp) ; 3.4.5.6. each for one single colony; 7. Negative control

Two-step enzyme digestion(Takara) for detection

![](https://static.igem.org/mediawiki/2015/5/55/NEFU_China_941806DA-0880-4966-B717-B339ABD5E15F.png)

![](https://static.igem.org/mediawiki/2015/f/f3/NEFU_China_BCB31D1A-B800-40BE-953A-AEBB127CFD69.png)

                     Fig 27. 1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1044bp) ; 3. digest vector (3167bp) ; 4.pcr product (1044bp)

The sequencing result is consistent with our designation.

![](https://static.igem.org/mediawiki/2015/4/4b/NEFU_China_CC2FDE7C-330B-4D09-B704-28B85F0C124A.png)

**4. pNZ8148-lsrD**

Miniprep (pEASY-T5 cloning vector with LsrD) with TIANprep Mini Plasmid Kit

**Insert lsrD**

1.PCR

![](https://static.igem.org/mediawiki/2015/7/72/NEFU_China_80F6A3BD-657A-497B-8AF4-970844664E23.png)

Double digestion (Takara)

![](https://static.igem.org/mediawiki/2015/b/b8/NEFU_China_23CDFBDF-A7D5-4660-AC6F-9AD84FB35942.png)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1[see manual](http://www.takara.com.cn/?action=Page&Plat=pdetail&newsid=101&subclass=1))

![](https://static.igem.org/mediawiki/2015/d/da/NEFU_China_149D0F60-9342-4910-BA55-2680DC1E313B.png)

Transformation [see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf)

Colony PCR

![](https://static.igem.org/mediawiki/2015/4/47/NEFU_China_8794F247-02E3-4D97-8041-C9A4E7CEFDA4.png)

![](https://static.igem.org/mediawiki/2015/1/15/NEFU_China_967B6351-6F71-4272-92AA-04BF6EF14561.png)

Fig 28. 1. Marker (DL2000) ; 2. Postive control (1000bp) ; 3.4.5.6. each for one single colony; 7. Negative control

Two-step enzyme digestion(Takara) for detection

![](https://static.igem.org/mediawiki/2015/3/3d/NEFU_China_3D3DE586-3ED2-4F91-830A-7DCB99A41AE8.png)

           ![](https://static.igem.org/mediawiki/2015/9/91/NEFU_China_944D1AE5-D310-462B-8277-B98E73168B66.png)

                      Fig 29. 1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1002bp) ; 3. digest vector (3167bp) ; 4.pcr product (1002bp)

The sequencing result is consistent with our designation.

![](https://static.igem.org/mediawiki/2015/f/f8/NEFU_China_61D0D2C3-BD0D-4854-91F8-16B186CD7B47.png)

**5. pNZ8148-lsrK**

Miniprep (pEASY-T5 cloning vector with LsrK) with TIANprep Mini Plasmid Kit

**Insert lsrK**

1.PCR

![](https://static.igem.org/mediawiki/2015/3/37/NEFU_China_95C9EA81-6633-48CA-BE0F-0C0C3FAB062B.png)

Double digestion (Takara)

![](https://static.igem.org/mediawiki/2015/d/d1/NEFU_China_288A9D71-88DA-425A-967D-D25D54FBC136.png)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1[see manual](http://www.takara.com.cn/?action=Page&Plat=pdetail&newsid=101&subclass=1))

![](https://static.igem.org/mediawiki/2015/1/16/NEFU_China_D51B611E-A3F0-4DBF-B64D-B108934C81A9.png)

Transformation [see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf)

Colony PCR

![](https://static.igem.org/mediawiki/2015/4/45/NEFU_China_C99CD053-9661-4A10-8CAE-3840AB97E4A8.png)

![](https://static.igem.org/mediawiki/2015/2/28/NEFU_China_2293F963-4AF7-44BB-8BC9-1F38560C6973.png)

Fig 30. 1. Marker (DL2000) ; 2. Postive control (1593bp) ; 3.4.5. each for one single colony; 6. Negative control

Two-step enzyme digestion(Takara) for detection

![](https://static.igem.org/mediawiki/2015/7/7a/NEFU_China_E2E5B4B4-135E-4DA6-BD61-430962B5DDFD.png)

                   ![](https://static.igem.org/mediawiki/2015/0/0c/NEFU_China_F7463EFC-3ABE-4F33-9ECA-32054C0D9F58.png)

Fig 31. 1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,1593bp) ; 3. digest vector (3167bp) ; 4.pcr product (1593bp)

The sequencing result is consistent with our designation.

![](https://static.igem.org/mediawiki/2015/c/c4/NEFU_China_A1799C0D-DD28-4664-AEDD-5B74934F7711.png)

**6. pNZ8148-lsrR**

Miniprep (pEASY-T5 cloning vector with LsrR) with TIANprep Mini Plasmid Kit

**Insert lsrR**

1.PCR

![](https://static.igem.org/mediawiki/2015/a/a0/NEFU_China_96E3CC7B-17FD-4DD8-9847-242EB7BB5266.png)

Double digestion (Takara)

![](https://static.igem.org/mediawiki/2015/6/60/NEFU_China_C393A81F-2AC5-40D6-A4FB-F47C14B6D3E6.png)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1[see manual](http://www.takara.com.cn/?action=Page&Plat=pdetail&newsid=101&subclass=1))

![](https://static.igem.org/mediawiki/2015/6/63/NEFU_China_D95E46ED-88DC-4C03-AAB8-8013D7CC1C3B.png)

Transformation [see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf)

Colony PCR

![](https://static.igem.org/mediawiki/2015/b/bf/NEFU_China_8B5B6280-B089-4BBD-B00C-3164FE9A3BF9.png)

![](https://static.igem.org/mediawiki/2015/8/8f/NEFU_China_49AC3B23-7060-47B0-99BA-CDA1E7955793.png)

Fig 32. 1. Marker (DL2000) ; 2. Postive control (960bp) ; 3.4.5.6.7.each for one single colony; 8. Negative control

Two-step enzyme digestion(Takara) for detection

![](https://static.igem.org/mediawiki/2015/3/30/NEFU_China_2E868823-CAF5-46C2-982C-1B5DB3420370.png)

          ![](https://static.igem.org/mediawiki/2015/5/58/NEFU_China_C8ED067B-D6B0-4AEB-9056-7F6286556087.png)

Fig 33. 1. Marker (DL10000) ; 2. Double enmyze digestion (3167bp,960bp) ; 3. digest vector (3167bp) ; 4.pcr product (960bp)

The sequencing result is consistent with our designation.

![](https://static.igem.org/mediawiki/2015/4/42/NEFU_China_A1DCD30B-0805-45AD-A384-11597A81AE1D.png)

**7. pHY300PLK-plsr-blue-T**

1. Molecular biology techniques: SOE (Splicing by overlap extension) PCR

**A. Primary PCR reaction**

**Segment1-plsr**

![](https://static.igem.org/mediawiki/2015/f/f3/NEFU_China_31952388-2D19-4FA8-8DBD-63EDA1E2CB4F.png)

**Segment2- Blue-T**

![](https://static.igem.org/mediawiki/2015/f/f7/NEFU_China_B4879185-4C26-4FC5-A133-5F5056B51D02.png)

![](https://static.igem.org/mediawiki/2015/f/f9/NEFU_China_FAFBCF0A-D050-461A-A4A2-825CD2A6B4E2.png)

Fig 34. 1. Marker (DL2000) ; 2. Blue-T (878bp) ; 3. plsr (250bp)

**B. Overlapping and elongation**

![](https://static.igem.org/mediawiki/2015/0/0b/NEFU_China_6F230DAC-C5D6-4BDB-88AA-02DA124DC03F.png)

**C. Second PCR reaction**

![](https://static.igem.org/mediawiki/2015/8/8d/NEFU_China_1E5EE16F-C0AB-4C36-85FB-F6082564BA8C.png)

                                                                                                                                            ![](https://static.igem.org/mediawiki/2015/9/9b/NEFU_China_1759504E-CD10-448E-A49A-81693C20EF31.png)

Fig 35. 1. Marker (DL2000) ; 2. pLsr-Blue-T (1128bp) ; 3. pLsr (250bp) ; 4. Blue-T (878bp)

Double digestion (Takara)

![](https://static.igem.org/mediawiki/2015/7/7d/NEFU_China_28CC959C-9C24-4F25-81A6-A7BD6F9AF770.png)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1[see manual](http://www.takara.com.cn/?action=Page&Plat=pdetail&newsid=101&subclass=1))

![](https://static.igem.org/mediawiki/2015/8/8a/NEFU_China_02420433-A28A-4085-AF17-0C250FF0A0ED.png)

Transformation [see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf)

Colony PCR

![](https://static.igem.org/mediawiki/2015/3/37/NEFU_China_6AFC9ED0-9BC4-48F2-BD1F-472ADF2A92F7.png)

![](https://static.igem.org/mediawiki/2015/6/67/NEFU_China_52E5F2E1-0F82-4B6D-91ED-87E7215CACF5.png)

Fig 36. 1. Marker (DL2000) ; 2.3. each for one single colony; 4. Negative control; 5. Postive control (1128bp)

Two-step enzyme digestion(Takara) for detection

![](https://static.igem.org/mediawiki/2015/4/47/NEFU_China_BAAEBCCD-55D3-49DD-9F51-442791AEC61B.png)

![](https://static.igem.org/mediawiki/2015/2/22/NEFU_China_2529692B-0C96-4516-AF67-92A828B120CE.png)

                  Fig 37. 1. Marker (DL10000) ; 2. Double enmyze digestion (4870bp,1128bp) ; 3. digest vector (4870bp) ; 4.pcr product (1128bp)

The sequencing result is consistent with our designation.

![](https://static.igem.org/mediawiki/2015/a/ad/NEFU_China_8D0E718D-241A-4FB4-8E21-B8F5AEFF7395.png)

**8. pBBR1MCS-5-lsrA**

**Insert lsrA**

Miniprep (pNZ8148 with LsrA) with TIANprep Mini Plasmid Kit

1.PCR

![](https://static.igem.org/mediawiki/2015/c/c2/NEFU_China_93E23CE5-9BC9-46AA-8B81-EB60A6154CBA.png)

Double digestion (Takara)

![](https://static.igem.org/mediawiki/2015/f/f9/NEFU_China_DBC8232D-3C4F-45A1-B117-880DD13B520E.png)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1[see manual](http://www.takara.com.cn/?action=Page&Plat=pdetail&newsid=101&subclass=1))

![](https://static.igem.org/mediawiki/2015/e/e1/NEFU_China_34C18741-B024-438E-A27C-68B7A2495375.png)

Transformation [see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf)

Colony PCR

![](https://static.igem.org/mediawiki/2015/f/fe/NEFU_China_11E19294-D9B6-46C5-8CCD-09991F733E28.png)

                                                                                                                 ![](https://static.igem.org/mediawiki/2015/8/83/NEFU_China_325A7E57-B20E-4848-838D-9BFD4B801241.png)

Fig 38. 1. Marker (DL2000) ; 2. Postive control (1952bp) ; 3.4.5.6.7. each for one single colony; 8. Negative control

Two-step enzyme digestion(Takara) for detection

![](https://static.igem.org/mediawiki/2015/c/cf/NEFU_China_8D7F0BEE-3055-456B-9200-6513B3EF7E05.png)

                                                                                                                    ![](https://static.igem.org/mediawiki/2015/8/80/NEFU_China_240B221E-02C5-4D9A-AA79-6CB566ED3546.png)

                  Fig 39. 1. Marker (DL10000) ; 2. Double enmyze digestion (4707bp,1952bp) ; 3. digest vector (4707bp) ; 4.pcr product (1952bp)

The sequencing result is consistent with our designation.

![](https://static.igem.org/mediawiki/2015/2/2b/NEFU_China_C5CA77A9-97DD-41B4-BE9C-38150F1BADEA.png)

**9. pBBR1MCS-5-lsrC**

Miniprep (pNZ8148 with LsrC) with TIANprep Mini Plasmid Kit

**Insert lsrC**

1.PCR

![](https://static.igem.org/mediawiki/2015/1/19/NEFU_China_67956AB6-06C7-4B1D-813A-C8C06B27DB44.png)

Double digestion (Takara)

![](https://static.igem.org/mediawiki/2015/1/1b/NEFU_China_F2FDDE95-1E32-4CDB-9271-A6446D7B41C4.png)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1[see manual](http://www.takara.com.cn/?action=Page&Plat=pdetail&newsid=101&subclass=1))

![](https://static.igem.org/mediawiki/2015/f/fc/NEFU_China_401376A0-12F4-4A49-B3D9-DD005FC17FA8.png)

Transformation [see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf)

Colony PCR

![](https://static.igem.org/mediawiki/2015/7/7f/NEFU_China_E94743DF-F79B-4D8E-BB03-E25EC77F9AF8.png)

                                  ![](https://static.igem.org/mediawiki/2015/7/7c/NEFU_China_61F8EE29-8DAA-4232-8A60-C3F36DAECF37.png)

Fig 40. 1. Marker (DL2000) ; 2. Postive control (1503bp) ; 3.4.5.6. each for one single colony; 7. Negative control

Two-step enzyme digestion(Takara) for detection

                                                                                                       ![](https://static.igem.org/mediawiki/2015/b/be/NEFU_China_3089B91F-DA4F-4575-99EC-6894AA28BD32.png)

                     Fig 41. 1. Marker (DL10000) ; 2. Double enmyze digestion (4707bp,1503bp) ; 3. digest vector (4707bp) ; 4.pcr product (1503bp)

The sequencing result is consistent with our designation.

![](https://static.igem.org/mediawiki/2015/6/69/NEFU_China_7F00C6E0-AEBB-40AF-8613-46A0CEA146BB.png)

**10. pBBR1MCS-5-lsrD**

Miniprep (pNZ8148 with LsrD) with TIANprep Mini Plasmid Kit

**Insert lsrD**

1.PCR

![](https://static.igem.org/mediawiki/2015/9/9d/NEFU_China_F478B484-1697-46AB-BE81-40B946C8C959.png)

Double digestion (Takara)

![](https://static.igem.org/mediawiki/2015/2/2b/NEFU_China_705F260A-117F-48E7-B075-D788F8C18E78.png)

Ligation (TaKaRa DNA Ligation Kit Ver.2.1[see manual](http://www.takara.com.cn/?action=Page&Plat=pdetail&newsid=101&subclass=1))

![](https://static.igem.org/mediawiki/2015/e/eb/NEFU_China_033F7B8F-50B3-460B-89FA-1A05F4AE9F53.png)

Transformation [see protocol](https://static.igem.org/mediawiki/2014/f/f5/NEFU_China_Transformation.pdf)

Colony PCR

![](https://static.igem.org/mediawiki/2015/1/1a/NEFU_China_0FCB7E53-DFE5-47A5-9837-C206326F6760.png)

Fig 42. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony (1439bp)

Two-step enzyme digestion(Takara) for detection

![](https://static.igem.org/mediawiki/2015/7/75/NEFU_China_566EB48F-EDC1-410B-B218-AEB3CC949CCC.png)

                            ![](https://static.igem.org/mediawiki/2015/6/6a/NEFU_China_40E915B9-202B-4ACC-BC28-7A308B5A28CF.png)

                    Fig 43. 1. Marker (DL10000) ; 2. digest vector (4707bp); 3. Double enmyze digestion (4707bp,1439bp) ; 4.pcr product (139bp)

The sequencing results are consistent with our designation.

![](https://static.igem.org/mediawiki/2015/3/3d/NEFU_China_83EAEDA1-43D8-4FAF-BDD4-BF689DB9ADE9.png)

**Transformation in *Lactobacillus***

**Electrotransformation of LsrBRK**

**Miniprep (pNZ8148–*LsrB*, pNZ8148–*LsrR*, pNZ8148–*LsrK*) with TIANprep Mini Plasmid Kit**

**1.Preparation of competent cells**

100 μl bacterial culture were inoculated into 50ml of MRS and incubated at 37°C. After overnight growth, a culture at the beginning of the stationary phase was harvested by centrifugation. The bacteria were washed three times with cold electroporation buffer (PB).The cells were then resuspended in PB to an OD600 of about 50.

**2.Electrotransfection**

100  μl of the cell suspension was mixed with 10 μl plasmid DNA(pNZ8148–*LsrB*, pNZ8148–*LsrR*, pNZ8148–*LsrK*). The sample was subjected to a 2.4kV, 200Ω, 25μF electric pulse in a 0.2cm cuvette.

**3.Recovery of transformants**

950 μl SMRS was immediately added, and the cells were incubated for 2 h at 37°C before they were plated on MRS supplemented with the appropriate antibiotic. The plates were incubated at 37°C for 2 to 3 days under anaerobic conditions.

**4.Clone identify**

![](https://static.igem.org/mediawiki/2015/e/e6/NEFU_China_2A32A9E4-41BC-4EF5-A396-74EA013FE6B9.png)

We repeated this transformation process for several times. Unfortunatelly, not a single colony was grown the plate.

Then we changed the parameters of electric pulse and tried several recovery medium, but we just got some unsatisfactory results.

![](https://static.igem.org/mediawiki/2015/a/ac/NEFU_China_F3EE6719-D8A6-4F55-B1CF-CC82059C567C.png)

Fig 44. 1. Marker (DL2000) ; 2.3.4.5.6.7 each for one single LsrB colony; 8.9.10.11.12.13 each for one single LsrR colony;

1. Marker (DL2000) ; 2.3.4.5.6.7 each for one single LsrKcolony

           ![](https://static.igem.org/mediawiki/2015/f/f0/NEFU_China_A6318DC8-2957-4C64-9061-6686F9255552.png)

Fig 45. 1. Marker (DL2000) ; 2.3.4.5.6.7 each for one single LsrB colony; 8.9.10.11.12.13 each for one single LsrR colony;

1. Marker (DL2000) ; 2.3.4.5.6.7 each for one single LsrKcolony

![](https://static.igem.org/mediawiki/2015/a/ad/NEFU_China_0C3ED3F4-8E71-4A6A-94CA-77035E009229.png)

Fig 46. 1. Marker (DL10000) ; 2.3.4.5.6. each for one single LsrB colony; 7.8.9. each for one single LsrR colony;

         1. Marker (DL10000) ; 2.3. each for one single LsrR colony; 4.5.6.7.8.each for one single Lsrk colony;

Finally, we realized it was the concentration of the plasmids which led to the low efficiency in transformation. We then did plasmid Maxiprep (see protocol) and concentration, and obtained plasmids in a concentraion of about 3000ng/μL. We also added 100μL denatured salmon sperm DNA to the cuvette before electroporation. This time, we eventually gained positive colonies.

**Clone identify**

![](https://static.igem.org/mediawiki/2015/a/a0/NEFU_China_85EBC823-DCA7-402D-9EAF-19C3ABD712A3.png)

Fig 47. 1. Marker (DL4000) ; 2.3.4.5.6. each for one single LsrB colony; 7. Postive control (1023bp) ; 8.9.10.11.12. each for one single colony ; 13. Postive control (1593bp)

           1. Marker (DL4000) ; 2.3.4.5.6. each for one single LsrR colony; 7. Postive control (960bp);

**Electrotransformation of *plsr+blue*pigment+T**

Maxiprep (see protocol) and concentration of pHY300PLK-pLsr- amilCP。

**Clone identify**

Colony PCR

![](https://static.igem.org/mediawiki/2015/1/12/NEFU_China_25F1499D-4D64-42D5-8B98-EA16935BE2C3.png)

![](https://static.igem.org/mediawiki/2015/0/03/NEFU_China_4E03A65D-FD94-4FCD-9E63-5EE7937F8FE5.png)

Fig 48.1. Marker (DL2000) ; 2. Postive control (1128bp) ; 3.4. each for one single colony; 5. Negative control

**Electrotransformation of pNZ9530 plasmid**

 The transformation procedures are similar to Electrotransformation of BRK

**Clone identify**

Colony PCR

![](https://static.igem.org/mediawiki/2015/a/a3/NEFU_China_809E57B0-5E52-436B-8B5E-CF02AEB4512F.png)

![](https://static.igem.org/mediawiki/2015/f/fa/NEFU_China_C0FC3573-F528-4E64-80D3-4E191EE3E45D.png)

![](https://static.igem.org/mediawiki/2015/8/8f/NEFU_China_C7602C12-5C38-4123-B391-0BABDCA7539A.png)

![](https://static.igem.org/mediawiki/2015/7/7d/NEFU_China_5F2D24C3-D76A-46F5-9A59-C1BCAB3A2271.png)

Fig 49: 1. Marker (DL2000) ; 2.3.4.5.6. each for one single colony (486bp) ; 7. Postive control

Generated by [Flight iGEM](https://2015.igem.org/Team%3ANEFU_China/Software).

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