Part:BBa_K1321334:Experience

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Applications of BBa_K1321334

This part was cloned into part BBa_K1321333 by Biobrick cloning, to yield BBa_K1321336. The destination vector, containing the AraC-pBAD regulatory elements, was linearized using SpeI and PstI and AcsAB (previously digested with XbaI and PstI, was ligated at a 1:1 using the T4 ligase. The ligation mix was transformed into chemically competent DH10B Escherichia coli and plated on LB Agar+Chloramphenicol plates, then incubated overnight at 37 degrees. 5 ml LB falcons supplied with 50 ug/ml Chloramphenicol were inoculated with a selection of freshly grown colonies for further restriction analysis (refer to Results for additional information).

The functionality of part BBa_K1321336 was assayed by inducing AcsAB-containing cells with 0.1% Arabinose in 5mL LB supplied with 1% Glucose and Chloramphenicol. Overnight incubations were set up at 30 °C and 37 °C, and both empty vector controls and un-induced controls were also assayed. During sonication procedures LB, soluble and non-soluble fractions (the latter dissolved in 1x PBS) were kept for further analysis. Congo Red at a concentration of 20uM was added to all samples, which were further incubated for 2h at static conditions and at room temperature to allow for Congo Red binding.

User Reviews

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