Notebook – Results – 1. Construction of BBa_J04450 + constitutive promoter/crr or cyaA

We planned to construct BBa_J04450 as circuit #1 (original circuit) followed downstream by our original circuit #2. Circuit #2 is composed by BBa_J2319 (constitutive promoter) / BBa_B0034 (RBS) / crr (IIA(Glc) or cyaA (Adenylate cyclase) / BBa_B0015 (double terminator).

So the first step was to clone either cyaA or crr from E. coli to Biobrick construct. PCR fragments with correct size were able to be obtained. After direct restriction with EcoRI and SpeI, the fragments were cloned into pSB1C3 backbone.

Next step was to construct, BBa_J2319 (chloramphenicol) / BBa_B0034 (ampicillin) and crr or cyaA (chloramphenicol) / BBa_B0015 (chloramphenicol). For BBa_J2319 / BBa_B0034 construction, prefix insertion was used. Thus ligation product was screened on ampicillin LB agar plate. While for crr or cyaA / BBa_B0015 construction, suffix insertion was used because the large size of crr or cyaA could be isolated by agarose gel excision. After BBa_J2319 / BBa_0034 (ampicillin) and crr or cyaA / BBa_0015 (chloramphenicol) constructs were obtained, prefix insertion was done to get BBa_J2319 / BBa_B0034 / crr or cyaA / BBa_B0015 in pSB1C3 backbone. Final step was prefix insertion of BBa_J00450 the original constructs.

In every steps of works, we only confirmed the insertion by colony selection in appropriate antibiotics containing LB agar plate, and purification of backbone plasmid for insertion by gel electrophoresis to completely separated small insert fragments. Thus in theory there will be no self-ligation. However for either prefix or suffix inserts, the digested samples were not purified, instead used directly for ligation reaction.

Below some representative gel electrophoresis data were shown.

Sponsors