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
Fig 1. 1. Marker (2000bp DNA Ladder) ; 2. Salmonella typhimurium 16s rRNA
2. Ligation (TaKaRa DNA Ligation Kit see protocol)
3. Transformation (see protocol)
4. Colony PCR
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.
The sequence was consist with that of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2.
B. Identification of Lactobacillus bulgaricus
1. PCR
Fig 3. 1. Marker (DL2000) ; 2. Lactobacillus bulgaricus 16s rRNA
2. Ligation (TaKaRa DNA Ligation Kit see protocol)
3. Transformation (see protocol)
4. Colony PCR
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
lsrB
lsrC
lsrD
lsrK
lsrR
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
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)
3.Transformation (see protocol）
4.Colony PCR
lsrA
Fig 7. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony (1536bp) ; 6.Negative control
lsrB
Fig 8. Clony PCR results. 1. Marker (DL2000) ; 2.3.4 each for one single colony (1023) ; 5.Negative control
lsrC
Fig 9. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony (1089bp) ; 6.Negative control
lsrD
Fig 10. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony (1002bp) ; 6.Negative control
lsrK
Fig 11. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony(1593bp) ; 6.Negative control
lsrR
Fig 12. Clony PCR results. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony; 6.Negative control
5.sequencing
Fig 13. Sequencing result of LsrA
Fig 14. Sequencing result of LsrB
Fig 15. Sequencing result of LsrC
Fig 16. Sequencing result of LsrD
Fig 17. Sequencing result of LsrK
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
Segment2-lsrD2
Fig 19. 1. Marker (DL10000) ; 1. LsrD1 ; 2. LsrD2
B. Overlapping and elongation
C. Second PCR reaction
Fig 20. 1. Marker (DL10000) ; 1. LsrD ; 2. LsrD1; 3. LsrD2
Ligation
Transformation (see protocol)
Clone pcr
Fig 21. 1. Marker (DL2000) ; 2.3.4.5.each for one single colony(1002bp) ; 6.Negative control
Sequencing
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
Double digestion (Takara)
Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)
Transformation see protocol
Colony PCR
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
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.
2. pNZ8148-lsrB
Miniprep (pEASY-T5 cloning vector with LsrB) with TIANprep Mini Plasmid Kit（see protocol）
Insert lsrB
1.PCR
Double digestion (Takara)
Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)
Transformation see protocol
Colony PCR
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
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.
3. pNZ8148-lsrC
Miniprep (pEASY-T5 cloning vector with LsrC) with TIANprep Mini Plasmid Kit（ see protocol）
Insert lsrC
1.PCR
Double digestion (Takara)
Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)
Transformation see protocol
Colony PCR
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
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.
4. pNZ8148-lsrD
Miniprep (pEASY-T5 cloning vector with LsrD) with TIANprep Mini Plasmid Kit
Insert lsrD
1.PCR
Double digestion (Takara)
Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)
Transformation see protocol
Colony PCR
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
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.
5. pNZ8148-lsrK
Miniprep (pEASY-T5 cloning vector with LsrK) with TIANprep Mini Plasmid Kit
Insert lsrK
1.PCR
Double digestion (Takara)
Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)
Transformation see protocol
Colony PCR
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
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.
6. pNZ8148-lsrR
Miniprep (pEASY-T5 cloning vector with LsrR) with TIANprep Mini Plasmid Kit
Insert lsrR
1.PCR
Double digestion (Takara)
Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)
Transformation see protocol
Colony PCR
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
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.
7. pHY300PLK-plsr-blue-T
1. Molecular biology techniques: SOE (Splicing by overlap extension) PCR
A. Primary PCR reaction
Segment1-plsr
Segment2- Blue-T
Fig 34. 1. Marker (DL2000) ; 2. Blue-T (878bp) ; 3. plsr (250bp)
B. Overlapping and elongation
C. Second PCR reaction
Fig 35. 1. Marker (DL2000) ; 2. pLsr-Blue-T (1128bp) ; 3. pLsr (250bp) ; 4. Blue-T (878bp)
Double digestion (Takara)
Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)
Transformation see protocol
Colony PCR
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
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.
8. pBBR1MCS-5-lsrA
Insert lsrA
Miniprep (pNZ8148 with LsrA) with TIANprep Mini Plasmid Kit
1.PCR
Double digestion (Takara)
Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)
Transformation see protocol
Colony PCR
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
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.
9. pBBR1MCS-5-lsrC
Miniprep (pNZ8148 with LsrC) with TIANprep Mini Plasmid Kit
Insert lsrC
1.PCR
Double digestion (Takara)
Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)
Transformation see protocol
Colony PCR
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
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.
10. pBBR1MCS-5-lsrD
Miniprep (pNZ8148 with LsrD) with TIANprep Mini Plasmid Kit
Insert lsrD
1.PCR
Double digestion (Takara)
Ligation (TaKaRa DNA Ligation Kit Ver.2.1see manual)
Transformation see protocol
Colony PCR
Fig 42. 1. Marker (DL2000) ; 2.3.4.5 each for one single colony (1439bp)
Two-step enzyme digestion(Takara) for detection
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.
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
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.
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
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
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
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
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
