Team:SCUT-China/Team/Notebook

May 29th to June 19th
1.Purchasing Erythromycin Streptomyces, activating, picking the fungus, and expanding genome. ( later proved to buy the wrong.)
2.Training and assisting to pick erythromycin streptomyces and extracting genome ( later proved to buy the wrong.)

July 6th to July 13th
1.Building docking domains (DD1,DD2,DD3), preparating competent cells named top 10. It was a shame that the establishment of docking domains have come to nothing because the new member made some mistakes.
2.Building the docking domains (DD4,DD5,DD6) unsuccessfully.

July 14th to July 21st
1. Building the docking domains ( DD1,DD2,DD3 ) again and achieving success.
2. Entrusting to Liang Yanhui to do the rest of establishment (DD4,DD5,DD6).
3. Trying a variety of methods for PCR, but failed.
4. The following are various systems of PCR we did during the period, however it failed because of the high GC content of the sequence.
5. Extracting genome of Saccharopolyspora erythraea NRRL 2338.
6. Structuring plasmid biobrick Docking Domain 4-prefix, 4-postfix and Docking Domain 6-prefix, 4-postfix successfully.
7. The size of target segment: about 200bp
The size of vector (pSB1C3): 2070bp
8. Verifying plasmid biobrick Docking Domain 5-prefix, 5-postfix, 1-prefix, 1-postfix and 3-prefix, 3-postfix.
9. The gradients of annealing temperature: 50,55,60

July 23rd to July 27th
1. Verifying plasmid biobrick Docking Domain 1-prefix, 1-postfix.
2. PCR gene clusters: eryAⅠ(11kb),eryAⅡ(11kb),eryAⅢ(9kb).
Primer pairs: DEBS1上,DEBS1下; DEBS2上,DEBS2下; DEBS3上,DEBS3下.
DNA polymerase: Primer STAR
The volume of total system: 10.0μl
Result: Failed
3. PCR gene clusters: eryAⅠ(11kb),eryAⅡ(11kb),eryAⅢ(9kb).
Primer pairs: eryAⅠ上, eryAⅠ下; eryAⅡ上, eryAⅡ下; eryAⅢ上, eryA Ⅲ下.
The volume of total system: 10.0μl
Result: Failed. Only got 2kb DNA segment.

July 28th to August 3rd
1. Cultivating new erythromycin streptomyces named NRRL2338 and extracting genome.
2. Helping others to complete the pcr domain and linker.
3. Doing a set of molecular experiment and transforming the docking domains into the pSB1C3 carrier successfully.
4. Mutant-enriched PCR
PCR gene clusters: eryAⅠ-500bp(12kb),eryAⅡ-500bp(12kb),eryAⅢ-500bp(10kb).
Primer pairs: eryAⅠ-500bp-上, eryAⅠ-500bp-下; eryAⅡ-500bp-上, eryAⅡ-500bp-下; eryAⅢ-500bp-上, eryAⅢ-500bp-下.
DNA polymerase: Primer STAR, GenSTAR, PCR Mix
1) The volume of total system: 10.0μl
Operation:
①idle cycle(without polymerase)
②94℃×2min→(98℃×10s→70℃×11min[1kb/min])×25Cycles→72℃×10min
2)The volume of total system: 10.0μl
Operation: 98℃×30s→(98℃×10s→60℃×10s→72℃×6min[2kb/min])×30Cycles→72℃×10min
3)The volume of total system: 10.0μl
2×PCR Mix: 5.0
Operation: 94℃×3min→(94℃×30s→60℃×30s→72℃×12min[1kb/min])×30Cycles→72℃×10min
Result: Failed. Got nothing.
5. First, we designed and synthesized primers of each gene segment, by which we amplified each segment then.

July 30th to August 3rd
1.Mutant-enriched PCR
PCR gene clusters: eryAⅠ-500bp(12kb),eryAⅡ-500bp(12kb),eryAⅢ-500bp(10kb).
Primer pairs: eryAⅠ-500bp-上, eryAⅠ-500bp-下; eryAⅡ-500bp-上, eryAⅡ-500bp-下; eryAⅢ-500bp-上, eryAⅢ-500bp-下.
The volume of total system: 10.0μl
Operation:
①idle cycle(without polymerase)
②94℃×2min→(94℃×30s→60℃×30s→70℃×7min)×10Cycles→(94℃×30s→60℃×30s→70℃×7min20s)×10Cycles→72℃×10min
Result: Failed. Got nothing.

2. Mutant-enriched PCR
PCR gene clusters: eryAⅠ-500bp(12kb),eryAⅡ-500bp(12kb),eryAⅢ-500bp(10kb).
Primer pairs: eryAⅠ-500bp-上, eryAⅠ-500bp-下; eryAⅡ-500bp-上, eryAⅡ-500bp-下; eryAⅢ-500bp-上, eryAⅢ-500bp-下.
The volume of total system: 10.0μl
Operation:94℃×2min→(94℃×30s→60℃×30s→70℃×7min)×10Cycles→(94℃×30s→60℃×30s→70℃×7min20s)×10Cycles→72℃×10min
Result: Failed. Got nothing.

3. Finding that Tet was out of effect, we amplified resistance gene again, replacing Tet with Zeocin. We achieved homologous recombination fragment by Fusion PCR. After that, we linked it with T vectors and transformed it into BL21(DE3).