Team:USP-Brazil/Notebook
Lab experiments
Our daily experiments involved the manipulation of E. coli, thus needing its cultivation. These bacteria was mainly grown in the lysogy broth (LB) following the stablished protocol for such medium. The LB agar plates were made adding 1,5% of bacto agar. Such medium were all incubated at 37°C. All the antibiotic concentration for strains harboring plasmids was followed as stated by the CLSI standards.
PCRs were performed utilizing either Taq DNA Polymerase (Sinapse Inc - P1011) for routine amplification or Q5® High-Fidelity 2X Master Mix (NEB - M0492S) for high-fidelity amplification. The reactions were performed as recommended by its protocol. All electrophoresis were performed in TBE 1x with 0,8% agarose gel at 90 volts.
Digestion and ligation reactions were all performed as stated by its protocols. We utilized mainly NEB enzymes for such experiments, following the molar ratio and concentration per enzymes units stated.
Bacterial transformation was performed either with electrocompetent cells for higher rate of transformants or chemically competent cells for routine transformations. Electrocompetent cells were accomplished through several washes with Milli-Q water and glycerol 10%, the transformation protocol were made at 1800V in an electroporation cuvette (1mm gap width). Chemically competent cells were prepared washing cells with divalent cations solutions (such as Mg2+ and Ca2+), the transformation protocol were made heat shocking the cells at 42°C.
Cell density were measured with a spectrophotometer and cell fluorescence were measured with SpectraMax Paradigm in a 96-well black plate to avoid neighbour well interference.
Two important experiments that need to be featured are:
Golden gate assay
Both approach A and approach B circuit were too big to be ordered in a single block. Because of that both IDT and Twist synthesis were fragmented and ordered in several blocks, which were linked through the NEB® Golden Gate Assembly Kit (BsaI-HF® v2), according to the Instruction Manual bellow.
Comparison efficiency test of pBAD and pLac promoters
These experiments was used in order to analysis the chemical promoters, pLac and pBAD, and the light promoter. During the experiment, all the promoters mentioned in the project were tested. Each one was grown in two types of media: 0.2% glicerol mineral media and 0.2% glucose mineral media. For pBAD different concentrations of arabinose were used and for pLac different concentrations of IPTG were used. All the experiments occurred overnight.
USP-Brazil
