Difference between revisions of "Part:BBa K4166004"

Revision as of 02:53, 12 October 2022

Camp +1 mutation.

Increased plasmid copy number. Increased P2 extension, stabilizes secondary structure in promoter.

Plasmid Copy Number and Expression - Alma 2022

We created a series of mutants of BBa_J04450 in the origin of replication, and wanted to both quantify the copy number and see the effect the copy number has on gene expression.

We isolated both genomic DNA and plasmid DNA from the cells and ran a serial dilution of these on a gel to observe the following.

From the intensity of these bands we estimate the copy number of this origin at ~307 copies/cell.

We found that, although this mutation increases plasmid copy number, it actually decreases expression. It could be due to collisions between DNA polymerase (attempting to replicate the plasmid) and RNA polymerase (attempting to transcribe the RFP gene).

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
23
INCOMPATIBLE WITH RFC[23]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
25
INCOMPATIBLE WITH RFC[25]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.

@@ Line 8: / Line 8: @@
 We created a series of mutants of BBa_J04450 in the origin of replication, and wanted to both quantify the copy number and see the effect the copy number has on gene expression.
+We isolated both genomic DNA and plasmid DNA from the cells and ran a serial dilution of these on a gel to observe the following.
+[[File:T--Alma--gel_004.png|700px|center]]
+From the intensity of these bands we estimate the copy number of this origin at ~307 copies/cell.
 [[File:T--Alma--pcnPlate.png|700px|center]]