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Team:Peking/Notebook/YWChen
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Yiwei Chen's Notes
Contents
July
7.26
PbrR MBP construction plan
August
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8.1
Prepare the plasmid DNA of T7+rbs+merT+rbs+merP+rbs+merC and PhiR73+Po promoter+rbs+antigen 43.
Prepare the plasmid DNA of 2-2E promoter+rbs+antigen 43.
Identify PhiR73+Po promoter+rbs+antigen 43 and 2-2E promoter+rbs+antigen 43 using electrophoresis.
8.2
Send the correct ones for sequencing.
Digest rbs+antigen 43 again.
8.4
Pick the clone of PhiR73+Po promoter+rbs+antigen 43 on the plate for autoaggregation assay.
Plan the assay.
Design new primers for antigen 43 which contains different promoters in the forward primer.
8.5
Preliminary autoaggregation assay.
Send PhiR73+Po promoter+rbs+antigen 43 for sequencing.
Transform PhiR73+Po promoter+rbs+antigen 43 into BL21 strains.
8.6
Pick the single clone.
Induce the antigen 43 gene’s expression by adding 10^-5 M IPTG for 4 hours.
Do the auto-aggregation assay( for detail, see the inductive aggregation page or antigen 43 part.)
PCR the rbs+agn43 using easyPFU.
Identify it using electrophoresis.
Digest the PCR product using EcoRl and Spel to put it into PSB1A2.
Do the ligation and transformation.
Send some of the PCR product for sequencing.
8.7
Digest merT+merC with Spel and Pstl, digest merP with Xbal and Pstl.
Retrieve the gel. Do the ligation.
Pick three clones of rbs+merP.
8.8
Prepare the plasmid DNA for rbs+merP and merT+merC.
Pick single clones of merT+merP+merC. Do the colony PCR.
Retrieve the rbs+agn43.
Do the ligation and transformation.
8.9
PCR antigen 43.
Nest PCR using nest primers. Do the first step.identify it using electrophoresis.
8.10
PCR antigen 43 using nest primers. Using different template.
Using better template to run PCR.
Do the transformation of antigen 43.
8.11
Retrieve the product of rbs+agn43. Digest it and do the ligation and transformation.
8.12
Pick the single clone of rbs+agn43.
Digest 1-23L with EcoRl and Xbal. Digest T7+merT+merP+merC with EcoRl and Spel.
Digest pmerT+GFP with Spel and Pstl, digest TPC with Xbal and Pstl.
Identify them using agarose gel electrophoresis.
Do the ligation and transformation.
8.13
Identify rbs+agn using electrophoresis.
Digest 1-23L with EcoRl and Xbal. Digest T7+merT+merP+merC with EcoRl and Spel.
Do the ligation and transformation.
8.14
Prepare the plasmid DNA.
Send the correct ones for sequencing.
8.15
See the sequencing result.
8.16
Digest PhiR73+Po promoter with EcoRl and Spel. Digest RBS with EcoRl and Xbal.
Retract the whole genome of K12 strain.
PCR antigen 43 using new template.
Pick single clones of antigen 43.
8.17
T7+TCP+Terminator done.
Transform it to BL21 strain.
Send antigen 43 for sequencing.
PCR antigen 43 using new template and better protocol.
Do the colony PCR of PhiR73+Po promoter+rbs.
Prepare the plasmid DNA and digest it for identification.
Do the ligation and transformation.
Digest pmerT+GFP with Spel and Pstl, digest TPC with Xbal and Pstl.
Digest pPbrA with Spel and Pstl. Digest rbs+T3 pol with Xbal and Pstl.
8.18
Retrieve the digested product .
Identify them using electrophoresis.
Pick the single clone of PCR of PhiR73+Po promoter+rbs, do the colony PCR.
Prepare the plasmid DNA.
Send the correct ones for sequencing.
Connect pPbra with rbs+T3 pol. Connect PhiR73+Po promoter+rbs with antigen 43.
Connect merp+GFP with TCP.
Do the ligation and transformation.
8.19
Identify them using electrophoresis.
Retrieve the gel.
Do the ligation and transformation.
PCR antigen 43. Digest it using EcoRl and Spel.
8.20
Pick the colony of PhiR73+Po promoter+rbs+antigen 43.
Put rbs+antigen 43 into PSB1A2 plasmid. Do the ligation and transformation.
Digest merp+GFP and T3 pol.
8.21
Identify the colony PCR product using electrophoresis.
Do the transformation.
Digest rbs+agn43 with EcoRl and Xbal. Digest PhiR73+Po promoter using EcoRl and Spel.
8.22
Do the autoaggregation assay of antigen 43.
Induce its expression by adding IPTG.
8.23
Do the autoaggregation assay.
8.25
Prepare the plasmid DNA for TPC+Terminator.
Send the correct ones for sequencing.
Construct pPbra using primers annealing.
8.26
Transform antigen 43.
8.27
Pick single clone of antigen 43.
Do the transformation of TPC+terminator.
Do the transformation of pPbrA.
Connect pBAD with rbs. Do the ligation and transformation.
8.30
Do the functional test of antigen 43.
Pick the clone of antigen 43.
Pick the clone of merP+GFP+TCP.
Do the ligation of pPbra+T3 pol again.
Pick the single clone of Pbad+rbs.
Do the transformation.
8.31
Send the correct merp+GFP and Pbad+rbs for sequencing.
Do the transformation.
Induce the expression of antigen 43.
September
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9.1
Connect the antigen 43 into plasmid.
Send merP+GFP+TCP for sequencing.
Connect pPbra+T3 pol using primers annealing.
Do the ligation and transformation.
9.2
Prepare the plasmid DNA for rbs+agn43.
See the sequencing result of merp+GFP+TCP.
Pick the clone of pPbra+T3 pol.
9.3
Send the rbs+agn43 for sequencing.
Digest rbs+agn43 with EcoRl and Xbal. Digest PhiR73+Po promoter with EcoRl and Spel.
Pick the clone of pPbra+T3 pol. Prepare the plasmid DNA.
9.6
Do the PCR of antigen 43 using Hifi Taq DNA polymerase.
Digest merP+GFP+TCP with EcoRl and Spel. Put it into PSB3K3 backbone.
Connect pPbra+T3 pol with 1-23L. pick the single clone. Prepare the plasmid DNA.
Do the ligation and transformation.
Pick the clone of Pbad+T3 pol.
Make competent cell containing pc+merR+merp+rbs+T3pol. Transform T3 promoter into it.
Induce the expression using different concentration of IPTG.
Re-suspend the cell. Measure the GFP intensity by a microplate reader.
9.7
Digest merP+GFP+TCP with EcoRl and Pstl to put it into PSB3K3.
Send pbra+T3 pol+terminator for sequencing.
Identify the pBAD+T3 pol using electrophoresis.
Make competent cells of pc+merR+merp+rbs+T3pol.
Transform T3 promoter into it.
9.8
Re-suspend the cell.
Measure the GFP intensity using microplate reader.
PCR antigen 43 using hifi Taq DNA polymerase.
9.9
PCR Rbs+agn 43 using agn for/rev primer to identify it.
Digest the correct ones using EcoRl and Spel.
Send the correct ones for sequencing.
Transform T3 promoter into cells containing pc+merR+merp+rbs+T3pol.
Send pBAD+T3 pol for sequencing.
pPbra+T3pol+terminator done.
Measure the GFP intensity using a microplate reader.
9.12
Digest rbs+agn43 with EcoRl and Xbal. Digest PhiR73+Po promoter with EcoRl and Spel.
Identify them using electrophoresis.
Retrieve the gel.
Do the ligation and transformation.
Pick the clone. ===9.13===. Digest Antigen 43 with EcoRl and Xbal. Digest PhiR73+Po promoter+rbs with EcoRl and Spel.
Identify them using electrophoresis.
9.14
PCR antigen 43.
Get the candidate of rbs+agn43.
Send the correct ones for sequencing.
9.15
Put antigen 43 into PSB1A2.
Do the ligation and transformation.
9.16
Digest PSB1A2 and PSB1A3.
9.17
Digest PSB1C3 with EcoRI and Spel.
Digest antigen 43 with EcoRl and Spel.
Do the ligation and transformation.
9.21
Retract the whole genome of K12 strain.
9.22
Nest PCR of antigen 43 using new template and new primers.
Put antigen 43 into PSB1C3.
Pick the single clone of antigen 43.
9.23
Prepare the plasmid DNA for antigen43.
Digest the plasmid DNA using EcoRl and Spel.
Identify them using electrophoresis.
Make the competent cell contains PBAD+ T3 pol. Transform T3 promoter+GFP into it.
Pick the clone of PMERT+T3 pol.
Digest PSB3C5 using EcoRl and Pstl.
9.24
PSB3K5: EcoRl and Pstl.
T7+TPC+Ter: Xbal and Pstl.
MerP+GFP:EcoRl and Spel.
Do the ligation and transformation.
9.25
Retrieve the digested merp+GFP.
Induce the expression of PBAD+T3 pol using 10^-5 M arabinose.
Do the antigen 43 PCR using touchdown PCR.
Do the ligation and transformation.
9.26
Make the competent cell contains T3 promoter+GFP. Transform pmert+T3pol and 1-18i+merR.
Do the colony PCR to identify it.
9.27
Attend the seminar.
9.28
Connect T7+PhiR73+Po promoter+rbs+antigen 43.
Do the ligation and transformation.
PCR the T3 promoter+PhiR73+Po promoter.
9.29
Retrieve the PCR product.
Digest the product using EcoRl and Spel.
Digest TCP with Xbal and Pstl. Identify it using electrophoresis.
Digest merp+GFP using Spel and Pstl.
Do the transformation.
9.30
Do the ligation and transformation.
Prepare the plasmid DNA and identify it using PCR.
October
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10.1
Digest pTET+T7 pol using EcoRl and Spel.
Retrieve the gel.
Connect T3 promoter+PhiR73+Po promoter+ antigen 43.
Do the ligation and transformation.
10.3
Connect pTET+T7 pol and T7 promoter+PhiR73+Po promoter+ antigen 43.
Do the ligation and transformation.
10.4
Do the auto-aggregation assay of antigen 43.
10.5
Do the auto-aggregation assay.
10.7
Digest merp+GFP using Spel and Pstl.
Digest TCP using Xbal and Pstl.
Do the ligation and transformation.
10.8
Identify the digested product using electrophoresis.
Do the ligation and transformation.
10.9
Prepare the plasmid DNA.
Identify them using PCR.
Send the correct ones for sequencing.
10.10
Antigen 43 clone step done.
Connect ptet+T7 pol with T7 promoter+PhiR73+Po promoter+ antigen 43.
Do the ligation and transformation.
10.11
Do the auto-aggregation assay.
10.12
Do the auto-aggregation assay.
10.13
Measure the result.
10.15
Attend group seminar.
Do the auto-aggregation assay.
10.16-10.21
Connect merp+GFP with TCP.
Connect pc+merR with terminator.
Transform these two plasmid into one single strain.
Antigen 43 auto-aggregation assay. Analyse the result.
10.21-10.25
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