Investigation of P3 threshold for E. coli resistance

• Plasmids were received (T18, ZHER2, LZT18, LZT25 and HER2 codon) and diluted. Also two pSB1C3 plasmids from iGEM containing RBS (BBa_E0020) and CFP (BBa_E0020) were diluted.
• Preparation of E. coli GB05 and set cultures overnight.
• Electroporation of the cells with the plasmids.
• Transfected cells were streaked out on chloramphenicol (Cm) and kanamycin (kan) plates.
• Colonies were picked and mixed with Cm and kan. These were stored in the fridge.
• Streaking out colonies from DHM1, BTH101 and K12 plates.

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Investigation of P3 threshold for E. coli resistance

• A miniprep was done with the seven cultures with the plasmids. A glycerol stock of each culture was saved.
• The concentration of the plasmids was measured using the nanodrop.
• A digestion of the plasmids was done and a gel was run.

Correct folding study of target protein

• pET28a vector preparation: digestion and dephosphorylation. Afterwards a gel was run and the DNA was extracted.
• A PCR was set to create HER2 with NheI and NotI restriction sites. A digestion was done and the product was run on a gel and purified. The concentration was measured with the nanodrop. This process was repeated some days later since the concentration was too low.

Investigation of P3 threshold for E. coli resistance

• 3 cultures for GB05 and K12 were set overnight.
• Transformation of T25 and gene III plasmids and promoter (pLac) in E. coli: gene III in GB05 and K12; T25 in GB05; promoter in K12 and GB05.
• Transformed colonies were plated on kan plates (gIII and T25) and Cm (pLac). A miniprep was done with the overnight cultures of pLac, T25 and gIII. The concentration of plasmid was measured with the nanodrop.
• New back-up cultures were set overnight.
• Cloning of P3 and RBS+CFP in pLac, followed by dephosphorylation and gel running and purification. The concentration was measured with the nanodrop.

Correct folding study of target protein

• Restriction digestion of HER2 and pET28a. The products were run on a gel and afterwards the weight was measured.

Investigation of P3 threshold for E. coli resistance

• Ligation of pLac + P3 and RBS + CFP: first set up the cultures for transformation, and the transform the electrocompetent cells: pLac in K12, pLac+P3 in K12 and GB05, and RBS+CFP in GB05. Plate the transformed cells and incubate them overnight.
• Plasmid miniprep from RBS+CFP in GB05 and pLac+P3 in GB05. The concentration of the plasmids was measured with the nanodrop. The products were then digested, dephosphorylated and loaded on a gel.

Conversion of BACTH into an iGEM standard and analysis of function

• Restriction digestion for the biobrick constructs and BACTH buildup: ZHER2, HER2, LZT25, LZT18, T18, T25 and P3.

Set up of flow system

• Preparation of the plates needed for the setup: one colony of pLac in K12 and pLac+pIII in K12 were streaked out and incubated in Cm+LB.