Difference between revisions of "Part:BBa K625005"

Latest revision as of 17:00, 1 October 2011

pSB6A5

This is the version 5 BioBrick vector backbone of the medium copy (minimal pBR322 origin) pSB6A1 plasmid. This means, that the cassettes for the ORI and antibiotic resistance have been minimized (reduction of the size and transcriptional terminators flanking the prefix and the suffix have been added, to trancriptionally insulate the inserted parts from the vector backbone machinery and vice versa. This minimization yielded a reduction in size from 4022 bp to 2743 bp.

For comparison of the copy number of the plasmids, triplicates of OD normalized bacterial cultures containing the respective plasmids were miniprepped. The DNA concentration was then determined by an agarose DNA gel electrophoresis of EcoRI digests of the purified plasmids (Figure 1). This experiment showed a clear reduction in copy number of the minimized version of the original plasmid (Figure 2). Possible reasons for this could be deletion of some unknown regulatory sequences during minimization of the ORI or transcription from within the insert into the origin region in psB6A1 impacting the replication machinery.

Figure 1: Agarose gel with 2-log-ladder loaded in in lane one, digests of triplicates of the EcoRI digests of pSB6A1 (lane 2-4) and pSB6A5 (lane5-7).

Figure 2: Agarose gel with 2-log-ladder loaded in in lane one, digests of triplicates of the EcoRI digests of pSB6A1 (lane 2-4) and pSB6A5 (lane5-7).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2722
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 2728
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2722
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found at 2722
Illegal suffix found at 2
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found at 2722
Plasmid lacks a suffix.
Illegal XbaI site found at 2737
Illegal SpeI site found at 2
Illegal PstI site found at 16
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal SapI site found at 771

@@ Line 5: / Line 5: @@
 This minimization yielded a reduction in size from 4022 bp to 2743 bp.
-For comparison of the copy number of the plasmids, triplicates of OD normalized bacterial cultures containing the respective plasmids were miniprepped. The DNA concentration was then determined by an agarose DNA gel electrophoresis of EcoRI digests of the purified plasmids (Figure 1). This experiment showed a clear reduction in copy number of the minimized version of the original plasmid (Figure 2).
+For comparison of the copy number of the plasmids, triplicates of OD normalized bacterial cultures containing the respective plasmids were miniprepped. The DNA concentration was then determined by an agarose DNA gel electrophoresis of EcoRI digests of the purified plasmids (Figure 1). This experiment showed a clear reduction in copy number of the minimized version of the original plasmid (Figure 2). Possible reasons for this could be deletion of some unknown regulatory sequences during minimization of the ORI or transcription from within the insert into the origin region in psB6A1 impacting the replication machinery.
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