Team:NCKU Tainan/Notebook
Notebook
Of Blood, Sweat and Tears
Construction of Pasr and PgadA
4/26~4/27
- PCR Pasr and PgadA from E. coli MG1655 chromosome. Confirmed and extracted products by DNA gel.
4/28
- Digested the pSB1C3-GFP plasmid. Ligase the plasmid with Pasr / PgadA and RiboJ by PCR. Confirmed by DNA gel electrophoresis. Transformed the gene in DH5 alpha and spread the bacteria on LB plates.
4/30~5/1
- Confirmed the transformation by colony PCR ,enzyme digestion and DNA gel electrophoresis. Selected the correct colonies for sequencing and cultured them on LB plates.
5/9
- Store the correct colonies in glycerol in -80°C.
6/1
- Transformed each plasmid into BL21(DE3)
The pH sensor test
5/9
- Prepared the M9 medium in different pH value.
5/11
- Pre-cultured the DH5 alpha-pSB1C3 -Pasr-sfGFP strain.
5/12
- Cultured the DH5 alpha-pSB1C3 -Pasr-sfGFP strain in M9 medium in different pH value. Measured the O.D. 600 value and fluorescence every hour.
5/18
- Pre-cultured DH5 alpha-pSB1C3 –PgadA-RiboJ-sfGFP strain.
- Prepared the M9 medium in different pH value.
5/19
- Cultured the DH5 alpha-pSB1C3 –PgadA-RiboJ-sfGFP strain in M9 medium in different pH value. Measured the O.D. 600 value and fluorescence every hour.
6/2~6/3
- Repeated the experiment on 5/11~5/12
7/29
- Pre-culture the BL21(DE3)-pSB1C3 –Pasr-sfGFP strain
7/30
- Cultured the BL21(DE3)-pSB1C3 –Pasr -sfGFP strain on 96 well plate in different pH value. Measure the fluorescence every 5 minutes in 30 minutes.
9/4
- Pre-culture the BL21(DE3)-pSB1C3 –PgadA-RiboJ-sfGFP strain
9/5
- Cultured the BL21(DE3)-pSB1C3 –PgadA-RiboJ-sfGFP strain strain on 96 well plate in different pH value. Measure the fluorescence every 5 minutes in 30 minutes.
Construction of CA
7/2
- PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis. Extracted the gene from DNA gel.
7/5
- PCR the CA. Confirmed by DNA gel electrophoresis and extracted the gene from DNA gel.
7/6
- Digested and ligase the pSB1C3 plasmid and CA gene.
7/10~7/11
- Repeated the PCR of CA, digestion, ligation.
7/12
- Transformed the pSB1C3-PT7-CA into DH5 alpha.
7/19
- Send the plasmid to sequencing
Construction of RbcL
7/2
- PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.
- Extracted the gene from DNA gel.
7/11
- PCR the RbcL with new primer.
7/20
- PCR the RbcL with old and new primer.
7/21~24
- Repeated the PCR of RbcL
8/1
- Ligate the RbcL with PLacI.
- Transformed the pSB1C3-PT7-RbcL and pSB1C3-PLacI-RbcL into DH5 alpha.
8/3
- Confirmed the pSB1C3-PT7-RbcL construct by colony PCR.
- Sent the gene to sequencing
Construction of RbcXS
7/2
- Prepared the M9 medium in different pH value.
7/3~7/21
- PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.
- Extracted the gene from DNA gel.
7/23
- PCR normal/mutant RbcXS with old and new primer.
7/24
- Confirmed by DNA gel.
Construction of PRK
7/2
- PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.
- Extracted the gene from DNA gel.
7/3~8/2
- PCR PRK.
8/1
- Ligated PRK with PLacI and PT7.
- Transformed into DH5 alpha.
8/2
- Confirmed by colony PCR. Selected the colony with correct length and sent to sequencing.
Whole construction
8/8
- Ligated PT7-CA with PLacI-PRK on pSB1C3
8/9
- Ligated PT7-RbcL with PT7-RbcXS on pSB1C3
8/28
- Inserted PT7-CA with PLacI-PRK into pSB3K3
SDS PAGE, Functional test and Total solution
7/25~7/30, 8/4~8/5, 8/11~8/12
- Ran SDS-PAGE of CA, normal-RbcXS
8/7, 8/9, 8/10
- Ran SDS-PAGE of RbcL and PRK
8/16
- Ran the SDS-PAGE of RbcL, RbcXS and PRK
8/18~8/19
- Activity test of CA
9/2
- Ran SDS-PAGE of pSB1C3-PT7-RbcL-PT7-RbcXS and pSB3K3-PLacI-PRK-PT7-CA
9/3
- Transformed pSB1C3-PT7-RbcL-PT7-RbcXS and pSB3K3-PLacI-PRK-PT7-CA into W3110
9/5~9/17
- Total solution.