Construction of P_asr and P_gadA

4/26~4/27

• PCR P_asr and P_gadA from E. coli MG1655 chromosome. Confirmed and extracted products by DNA gel.

4/28

• Digested the pSB1C3-sfGFP plasmid. Ligase the plasmid with P_asr / P_gadA 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 -P_asr-sfGFP strain.

5/12

• Cultured the DH5 alpha-pSB1C3 -P_asr-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 –P_gadA-RiboJ-sfGFP strain.
• Prepared the M9 medium in different pH value.

5/19

• Cultured the DH5 alpha-pSB1C3 –P_gadA-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 –P_asr-sfGFP strain

7/30

• Cultured the BL21(DE3)-pSB1C3 –P_asr -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 –P_gadA-RiboJ-sfGFP strain

9/5

• Cultured the BL21(DE3)-pSB1C3 –P_gadA-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-P_T7-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 P_LacI.
• Transformed the pSB1C3-P_T7-RbcL and pSB1C3-P_LacI-RbcL into DH5 alpha.

8/3

• Confirmed the pSB1C3-P_T7-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 P_LacI and P_T7.
• 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 P_T7-CA with P_LacI-PRK on pSB1C3

8/9

• Ligated P_T7-RbcL with P_T7-RbcXS on pSB1C3

8/28

• Inserted P_T7-CA with P_LacI-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-P_T7-RbcL-P_T7-RbcXS and pSB3K3-Plac-PRK-P_T7-CA

9/3

• Transformed pSB1C3-P_T7-RbcL-P_T7-RbcXS and pSB3K3-Plac-PRK-P_T7-CA into W3110

9/5~9/17

• Total solution.