Difference between revisions of "Part:BBa K2491030"

Latest revision as of 03:56, 28 October 2017

RK2 Broad Host Range Vector with Kanamycin Resistance

The goal of this improvement was to replace p15a origin of replication in pSB3K3 (https://parts.igem.org/Part:pSB3K3) with the RK2 ori to generate a shuttle vector as a broad host range vector (i.e. can replicate in E.coli and other microorganisms).

The RK2 origin of replication is a broad-host-range plasmid belonging to the incP incompatibility group that can be maintained in a large number of bacteria. The minimal region for replication and maintenance consists of an origin of replication, oriV, and the plasmid replication initiator protein TrfA protein, that activates oriV. The copy number of RK2 is about 4-7 per cell in E. coli, 3 in Pseudomonas aeruginosa, and 4-7 in Agrobacterium

Proof Of Concept

Clone verification. The following primers annealing in the TRFA region of the RK2 backbone vector and the kanamycin region were used to verify the construct by amplification and sequencing of the backbone: TRFA_F: 5’-CCGACGGATGTTCGACTATTT-3’, TRFA_R: 5’-AGTAAAGCGCTGGCTGAA-3’, KM_F: 5’-AAAGCCGTTTCTGTAATGAAGG-3’, and KM_R: 5’-CCTGTTGAACAAGTCTGGAAAG-3’. The data showed that the 2 regions were present. In addition, cultures containing the RK2 backbone harboring the RFP gene [biobrick part BBa_K2491030 containing biobrick part BBa_J04450] showed a red pellet (right vial) compared to a control (left vial) without the RFP gene.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal prefix found at 1508
Illegal EcoRI site found at 3141
Illegal XbaI site found at 3156
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 1508
Illegal EcoRI site found at 3141
Illegal NheI site found at 1384
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 1514
Illegal NotI site found at 3147
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 1508
Illegal EcoRI site found at 3141
Illegal XhoI site found at 177
Illegal XhoI site found at 1020
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found at 1508
Illegal suffix found at 2
Illegal EcoRI site found at 3141
Illegal XbaI site found at 3156
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found at 1508
Plasmid lacks a suffix.
Illegal EcoRI site found at 3141
Illegal XbaI site found at 1523
Illegal XbaI site found at 3156
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NgoMIV site found at 1552
Illegal NgoMIV site found at 2143
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.

@@ Line 6: / Line 6: @@
 The RK2 origin of replication is a broad-host-range plasmid belonging to the incP incompatibility group that can be maintained in a large number of bacteria. The minimal region for replication and maintenance consists of an origin of replication, oriV, and the plasmid replication initiator protein TrfA protein, that activates oriV. The copy number of RK2 is about 4-7 per cell in E. coli, 3 in Pseudomonas aeruginosa, and 4-7 in Agrobacterium
+== Proof Of Concept ==
+Clone verification. The following primers annealing in the TRFA region of the RK2 backbone vector and the kanamycin region were used to verify the construct by amplification and sequencing of the backbone: TRFA_F: 5’-CCGACGGATGTTCGACTATTT-3’, TRFA_R: 5’-AGTAAAGCGCTGGCTGAA-3’, KM_F: 5’-AAAGCCGTTTCTGTAATGAAGG-3’, and KM_R: 5’-CCTGTTGAACAAGTCTGGAAAG-3’. The data showed that the 2 regions were present. In addition, cultures containing the RK2 backbone harboring the RFP gene [biobrick part BBa_K2491030 containing biobrick part BBa_J04450] showed a red pellet (right vial) compared to a control (left vial) without the RFP gene.
+[[Image:Red_Gene_for_RK2.png|600px]]