The Gas Vesicle gene cluster (BBa_I750016) submitted by Melbourne 2007 team and the LacI and CAP regulated promoter BBa_R0010 were combined, and the product ligated into vector pSB1AC3. The construct was used to test and caracterize the gas vesicle formation.
Melbourne GVP cluster was used containing the 400bp repeat in gvpL
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and this regulator. This part is incompatible with environments containing lactose or lactose analogs.
This 5.7Kbp part contains 11 open reading frames coding for gas vesicle genes (gvpB,R,N,F,G,L,S,K,J,T and U) from Bacillus megaterium VT1660 AF053765. Original AF053765 DNA in pBluescriptIIKS plasmid (pNL29 in "Gas Vesicle genes identified in Bacillus megaterium and Functional Expression in Escherichia coli, Ning Li and Maura C. Cannon Jounal of bacteriology May 1998 p2450-2458).
This 5.7Kbp part contains 11 open reading frames coding for gas vesicle genes (gvpB,R,N,F,G,L,S,K,J,T and U) from Bacillus megaterium VT1660 AF053765. Promotion of the sequence results in expression of gas vesicles which slow the rate of settling of the cells in Saline.
Site directed mutagenesis was performed in four rounds to remove 3 PstI sites and one EcoRI site from gvpL in the sequence. Primers with biobrick prefix and suffix were then used in a PCR reaction to amplify the part. Finally BBa_J61035 was used to provide the plasmid backbone which introduced a RBS at the front of the part.
10 repeats of 40bp induced durring mutagenesis
Conversion of the iGEM parts registry to SBOL2.1