Wiki/Team:SJTU-BioX-Shanghai/Notebook/log3

September 1st, 2012

Preparing parts

Reconstructed plasmid

• pSB1C3

Parts:

• Dsb-Lactmase
• FL3-Lgt
• LLG(DsbLac-fLgt-fGFP)
• rbs-TesA
• rbs-FabG
• rbs-FabI
• rbs-FabZ

Process

1. 1. PCR amplify the insert fragments
   2. gel-purification
   3. ligation
   4. transformation

System

PCR system:

Enzyme	2μl
10×buffer	2μl
Template	≤2μl
dNTP	10μl
Mg2+	6μl
primerF/R	2μl/2μl
Sterile water	up to 100μl

September 2nd, 2012

Preparing parts

Making sure and amplifying colony for plasmid extraction

Process

1. 1. check the plates
   2. pick single colonies
   3. colony PCR for certification
   4. incubation

ATTENTION:

Add chloramphenicol into the culuture.

September 3rd, 2012

Preparing parts

Plasmid extraction and certification

Process

1. 1. Plasmid extraction
   2. Enzyme digestion to make certification
   3. Keep the right constructed parts

System

Enzyme digestion system fro certification

Enzyme(EcoR1/Pst1)	0.1μl/0.1μl
10×FD Buffer	0.5μl
Template	≤1μg
Sterile water	up to 5μl

September 4th, 2012

Transform triple plasmids together

We need to try and make sure that triple plasmids can stay well in a same cell.

Plasmids

• M1FA:VEGC:pET
• M2FB pRSF
• M3FE:VED:pACYC

Process

1. 1. Transform triple plasmids into BL21
   2. Spread onto the plates

September 5th, 2012

Check triple plasmids system and make ligation

• The triple plasmids cell grows
• Colony PCR certification
• incubation for plasmid extraction
• Make triple plasmids ligation

New ligation

1. 1. pET: FabG,FabI
   2. pACYC: TesA

September 6th, 2012

Preparing parts

Reconstructed plasmid

• pSB1C3

Parts:

• M1-TesA
• M2-FabG
• M3-FabZ
• M4-FabI
• M1-EGFP1
• M2-EGFP2
• M3-EGFP1
• M4-EGFP2

Process

1. PCR amplify the insert fragments
2. gel-purification
3. ligation
4. transformation(TOP10)
5. spreading

September 7th, 2012

Preparing parts

Making sure and amplifying colony for plasmid extraction

Process

1. 1. check the plates(red colonies are not right)
   2. pick single colonies
   3. colony PCR for certification
   4. incubation for plasmid extraction

ATTENTION:

Add chloramphenicol into the culuture.

September 8th, 2012

Membrane switch construction

construction of plasmid pACYCDuetM-1,pETduetM-1

Process

structure of these plasmids needed to be constructed

• pACYCDuetM-1:vioB， vioE(site1,site2)
• pETduetM-1:vioA，vioC(site1,site2)

ATTENTION:

The different order of the two insert fragments ligated to the plasmid may make a little difference.

September 9th, 2012

Preparing parts

Reconstructed plasmid

• pSB1C3

Parts:

• fl3_1EGFP
• fl3_2EGFP
• MA1_VioA
• MA2_VioB
• MA3_VioE
• MA4_VioC

Process

1. PCR amplify the insert fragments
2. gel-purification
3. ligation
4. transformation(TOP10)
5. spreading

September 10th, 2012

Preparing parts

Redo some plasmids that failed the days before and make certification of yesterday's parts

Process

• redo parts that failed the days before

• make certification

1. 1. check the plates(red colonies are not right)
   2. pick single colonies
   3. colony PCR for certification
   4. incubation for plasmid extraction

September 11th, 2012

Membrane Accelerator

Fatty acids system construction.

Process

• plasmids construction: TesA;FabG;FabI;FabZ

• transform and test the fatty acids result.

• compare with the wild type and the constructed type

September 12th, 2012

Membrane protein polymerization test

Test for the polymerization of the membrane parts.

Process

• membrane polymerize parts:

0、Lgt-E1&Lgt-E2 1、M1-E2&M2-E2
2、M2-E2&M3-E1   3、M3-E1&M4-E2
4、M3-E1&VM4-E2  5、VM4-E2&VM5-E1

• Culture the cell containing these parts.

• Test for EGFP.

September 13th, 2012

Preparing parts

Reconstructed plasmid

• pSB1C3

Parts:

• RNA－D0
• FL3-Lgt
• Rbs_dsbAss_pdz_domain
• Fl3_gbd_domain
• Rbs_dsbAss_vvd_wildtype

Process

1. ligation
2. transformation(TOP10)
3. spreading

September 14th, 2012

Preparing parts

make certification of yesterday's parts

Process

• redo parts that failed the days before

• make certification

1. 1. check the plates(red colonies are not right)
   2. pick single colonies
   3. colony PCR for certification
   4. incubation for plasmid extraction

September 15th, 2012

Fermentation

Fatty acids system fermentation.

Process

• culture the cells

• extraction of products.

• running GC -MS for products test

September 16th, 2012

Ligation

Ligate M3-f with Vio C Ligate M2-f with Vio B

Ligation System

10X T4 DNA Ligase Buffer 	 2 μl 
Vector DNA 	 0.025 pmol 
Insert DNA 	 0.076 pmol 
T4 DNA Ligase 	 1μl 
Nuclease-free water 	 to 20 μl

Digestion identification

Ecor1, Past 1

Enzyme(EcoR1/Pst1)	0.1μl/0.1μl
10×FD Buffer	0.5μl
Template	≤1μg
Sterile water	up to 5μl

September 17th, 2012

Transformation

Transform M4-F-C, M2-F-B 1ul each into 30ul competent cells.

September 19th, 2012

Ligation

Ligate M3 to pUC19 Ligate M2D to pRSF Ligate M2B to pUC19

system

10X T4 DNA Ligase Buffer 2 μl

Vector DNA 0.025 pmol

Insert DNA 0.076 pmol

T4 DNA Ligase 1μl

Nuclease-free water to 20 μl

Digestion identification

Ecor1, Past1

system

• Enzyme 1μl
• 10×FD Buffer or FD Green buffer 2μl
• DNA ≤1μg
• Sterile water up to 20μl

September 18th, 2012

Plasmid extraction

PCR identification

• Reaction temperature:

            Three-step method：
        Predenature
          94℃，2min
        Denature，annealing and extension
          98℃ 10sec   
         {
         （56）℃  30sec
          68℃  30sec/kb
          }
                       × 34 cycles
          4℃

September 19th, 2012

Ligation

Ligate M3 to pUC19 Ligate M2D to pRSF Ligate M2B to pUC19

system

10X T4 DNA Ligase Buffer 2 μl

Vector DNA 0.025 pmol

Insert DNA 0.076 pmol

T4 DNA Ligase 1μl

Nuclease-free water to 20 μl

Digestion identification

Ecor1, Past1

system

• Enzyme 1μl
• 10×FD Buffer or FD Green buffer 2μl
• DNA ≤1μg
• Sterile water up to 20μl

September 20th, 2012

Transformation

Transform M1-TesA with M2-FabG, M3-FabZ with M4-FabI 1ul each into 30ul competent cells.

Ligation

VVD(c) with pET-Duet

VVD(d) with pACYC-Duet

Ligation System

10X T4 DNA Ligase Buffer 	 2 μl 
Vector DNA 	 0.025 pmol 
Insert DNA 	 0.076 pmol 
T4 DNA Ligase 	 1μl 
Nuclease-free water 	 to 20 μl

September 21st, 2012

Colony Picking

M1-TesA with M2-FabG, M3-FabZ with M4-FabI separately each pick up 6 colons. Incubate them into 5ml LB.

Transformation

VVD(c)-pET-Duet, VVD(d)-pACYC-Duet each into 30ul competent cells.

September 22nd, 2012

Fermentation

M1-TesA with M2-FabG, M3-FabZ with M4-FabI for 24hours

Digestion identification

VVD(c)-pET-Duet, VVD(d)-pACYC-Duet

Enzyme	1μl
10×FD Buffer or FD Green buffer	2μl
DNA	≤1μg
Sterile water	up to 20μl

September 23rd, 2012

Fig9.23 GCMS results

GC-MS

used GC-MS to quantify the total amount of fatty acid.

Ligation

Vio A with pET-Duet

Vio B with pACYC-Duet

Ligation System

10X T4 DNA Ligase Buffer 	 2 μl 
Vector DNA 	 0.025 pmol 
Insert DNA 	 0.076 pmol 
T4 DNA Ligase 	 1μl 
Nuclease-free water 	 to 20 μl

September 24th, 2012

Fig 9.24 GCMS results

GC-MS

used GC-MS to quantify the total amount of fatty acid from the bacteria.

Digestion identification

Vio A with pET-Duet, Vio B with pACYC-Duet

Enzyme	1μl
10×FD Buffer or FD Green buffer	2μl
DNA	≤1μg
Sterile water	up to 20μl

September 25th, 2012

GC-MS

Data Analysis

Sequencing identification

Transformation

Transform pET-Duet, pACYC-Duet 1ul each into 30ul competent cells.

September 26th, 2012

Colon picking

pET-Duet, pACYC-Duet each pick 6 colons Incubate them into 5ml LB.

Induction

Induce the bacteria with cofactors and incubate them under the blue light

HPLC

Analysis the violacein pathway products