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).

August 4th to August 11th
1. Acquiring PT7+RBS and TT7 from the flats of iGEM, transforming to pSB4A5, augmenting and extracting the plasmid for employs. (but later we proved there was a problem. )
2. Compounding the pccB and accA.
3. Gaining birA by PCR and transforming it into the pSB1C3 carrier.
4. PCR of prpR, prpE and sfp
5. Mutant-enriched PCR
Result:
A. failed. Got nothing.
B. Got 5kb segment
C. Got 5kb segment
6. We extracted plasmids and amplified homologous recombination fragments with plasmids above as template. Meanwhile, we redesigned two pairs of primers. The two pairs of primers were designed to amplify a fragment with upriver homologous arm as long as 700bp and downriver homologous arm as long as 1600bp, and a fragment with upriver and downriver homologous arm both as long as 600bp, in order to increase gene knock-out successful rate. Then, we tried to conduct gene knock-out.

August 12th to August 19th
1. After the completion of the pccB and accA，starting to build the construction of RBS+pccB and RBS+accA.
2. Starting the preparation of TT7 afresh.
3. Building the construction between PT7+RBS and birA. The determination of the sequence and the transform of the pSB4A5 were proved successful.
4. Built “KS-AT1 + -KR1, KS-AT2 +-KR1, KS-AT1 + -ACP-, KS-AT2 + -DH-ER-” successfully.The construction of pT7-RBS and loading domain failed.
5. Construction :ACP1+KS-AT2 and KR2+ACP2
6. Construction: KR1+ACP2 and KR2+ACP2
7. Gene knock-out without PKD46 has been conducted 3 times overall. Using PCR as dictation, we found only one bacterial strain up to standard. By the way, we achieved PKD6, so we transformed PKD46 into BL21(DE3) and prepared competent E.coli with PKD46.

August 20th to August 27th
1.Building the downstream of docking domains (DD1,DD2,DD3,DD4,DD6) and the third module, starting to assemble the pSB4A5.
2.Building the construction among PT7+RBS+birA, RBS+accA2 and RSB+pccB+PT7. ( Later, we proved it was wrong because of the false PT7 .)
3.Finished the construction of (KS-AT1)+（-KR1）+（-ACP）+(KS-AT2) , (KS-AT2)+（-DH-ER- ) +(KR1)+（-ACP), (pT7-RBS) + (loading domain) successfully. Construction of the (KS-AT2)+（-KR1）+（-ACP）+(KS-AT2), (KS-AT2)+（-DH-ER-）+ (KR2)+（-ACP) failed.
4.Construction: ACP1-KS-AT2+KR1-ACP2 and ACP1-KS-AT2+KR2-ACP2
5.Construction: ACP1-KS-AT2-KR1-ACP2+TE-TT7 and ACP1-KS-AT2-KR2-ACP2+TE-TT7
6.A bacterial strain up to standard was achieved when we conducted gene knock-out with PKD46 for the first time. Meanwhile, as identification result showed, frameshift mutation had taken place for sfp gene, so we have to transform sfp gene into E.coli.

August 28th to August 31th
1.Finishing the fabrication of pSB4A5.
2.Finished the consturction of DEBS1 and DEBS1’ successfully.

September 1th to September 7th
1.Finishing the verification of correctness and the method of representation.
2.The modification of E.coli. Removed pKD46 from BL21(DE3) unsuccessfully. Because BL21 (DE3) had Zeocin resistance genes and pKD46 with Amp resistance genes, if it is eliminated successfully, BL21(DE3)shouldn’t grow in the plate with Amp. However , BL21(DE3) grew in the plate with Amp.
3.We designed the primers of sfp and amplified sfp. After that, we linked it with another plasmid of other parts in our experiments.

September 8th to September 15th
1.Finishing all kinds of expressions.
2.Because the expression of product failed, we had to verify the previous DEBS1 and DEBS1’ again and prepared to reconstruct the loading domain+module1 + module2.
3.SDS-PAGE electrophoresis: prpE, DEBS1+TE, sfp

September16th to September 25th
1.Reconstructed DEBS1 and DEBS1’and finished the construction of loading domain +module1+module2 successfully.

September 26th to October 1th
1. Finished the construction of DEBS1 and DEBS1’.
2. Constructed the pT7-RBS + OFP successfully.

October 2th to October 8th
1 .Constructed the pT7-RBS+OFP + loading domain+ module1+module2 unsuccessfully.
2. Finished the construction of pT7-RBS+RFP +loading domain+ module1+module2 successfully prepared for the fluorescent detection later.
3. Took part in the expression experiment and started to express for 7.1 and 8.3.

October 9th to October 17th
1. Induced the expression of 13-1,13-2,14-1,14-2, 14-3 and detected the product.