Team:Amazonas-Brazil/Notebook
MAY
(05.31) -Late Registration is done.
JULY
(07.03) -Our iGEM box was detained in Brazilian Customs
AUGUST
Week 0 (08.05-08.12)
-Our Twist’s sequence was detained in Brazilian Customs
-Plasmidial extraction of EcN
-Stock preparation of EcN
-Trial of Confirmation of EcN strain
Week 1 (08.19-08.26)
-Our IDT sequences arrived safely!
-Arrived parts were cloned in E.coli DH5alpha and streaked
-Biobricks Inoculation
-Miniprep of BioBricks
Week 2 (08.26-09.02)
-Analytical Digestion of Secretion System Biobricks
-Purification, quantification and Ligation of Secretion System Biobricks
-Transformation and Inoculum of Secretion System Biobricks
-Digestion of Biobricks
-Plasmid extraction for confirmation of cloning.
-Blue White screening of Secretion System
SEPTEMBER
Week 3 (09.02-09.09)
After dealing with customs, our Twist Bioscience sequences arrived safely!
-Blue White screening
-Analytical digestion of Secretion System and plasmidial extraction
-Inoculation and Eletrocompetence of JM110
-Plate streaking of EcN and DH5alpha
-Digestion of Twist parts
-Digestion of EcN Cryptid plasmids (pMUT1 and pMUT2)
-MiniPrep of parts
-Quantification of parts by QuBit
Week 4 (09.09-09.16)
-Precipitation
-Plasmidial extraction
-PCR and colony PCR
-Digestion of part from USP and from secretion system
-Inocule of EcN and DH5alpha
-Purification, quantification and ligation of parts from secretion system
Week 5 (09.16-09.23)
-Quantification of secretion system by NanoDrop
-Ligation of Secretion system biobricks
-Precipitation of secretion system
-Digestion of Secretion system
-Gblock Purification
-Transformation of system on DH5alpha
-Inocule of JM110 with secretion system
-Plating of DH5alpha
Week 6 (09.23-09.30)
-Transformation of secretion system
-Digestion of secretion system
-Quantification of gBlocks
-Ligation of gBlocks to pUC19 vector
OCTOBER
Week 7 (09.30-10.07)
-Precipitation of ligation of gBlock7 to TolC_B0015_pSB1C3
-Transformation of the ligation in DH5alpha
-Cloning of Secretion and AND gate modules in EcN and DH5alpha
-InPreparation of backbones and acquiring of already made backbones
-Trial of validation of AND gate
Week 8 (10.07-10.14)
-Padronization of Growth curves
-Trial of Validation of AND gate and secretion module
-Cloning of Secretion and AND gate modules in EcN and DH5alpha
-Inocules of EcN
-Preparation of DH5alpha electrocompetent and Chemocompetent
-Preparation of EcN electrocompetent and chemocompetent
Week 9 (10.14-10.21)
-Preparation of EcN electrocompetent cells
-Transformation of RFP_pSB1C3, GFP_pSB1C3, gblock3_pUC19, gfpmut3_pUC19, ANDgate_pUC19 in E coli Nissle
-Cell lyses, protein dosage and SDS-Page of E coli Nissle, gBlock3_pUC19 in EcN, ANDgate_pUC19 in EcN and gfpmut3_pUC19 in EcN
-Growth curve of E coli Nissle with the plasmids GFP_pSB1C3, RFP_pSB1C3, GFPmut3_pUC19, gBlock3_pUC19, pUC19 and ANDgate_pUC19.
-Characterization of ANDgate_pUC19 with hypoxia and lactate
