Part:BBa_K1585351
glgP targeting plasmid
This plasmid is targeting glgP in the E. coli genome by expressing two sgRNAs for CRISPR/Cas9 genome editing. The complete glgP coding sequence is cut out and multiple stop codons are introduced by the repair templates.
Usage and Biology
The construct is part of a two-plasmid knockout system published by [http://aem.asm.org/content/81/7/2506.long| Jiang et al]. This part, the pTargetT, contains the sgRNA to target the glgP coding sequence. Additionally, the construct consists of a repair template with two 400 bp homology arms for homology-directed repair. To succesfully knock out the glgP, you need a second plasmid, pCas. It contains the pCas gene as well as a temperature-sensitive repA101ts ori. To increase recombination efficiency in E. coli, it also expresses lambda-Red genes behind an inducible arabinose-promoter. Upon induction with IPTG, a sgRNA is transcribed to cut the pMB1 ori of the pTargetT. After cloning both the targeting plasmid and the pCas plasmid in BL21 Gold (DE3) and inducing with arabinose, the Cas9 gene is expressed. Guided by the sgRNA, it cleaves the double strand at the beginning of the glgX gene. The succesful genome editing could be verified by sequencing.
Sequencings of the respective genomic region have shown that glgP was successfully cut out. In further experiments, the effect of this gene knockout was characterized. The expected consequence was an accumulation of glycogen due to the absense of a glycogen phosphorylase degrading alpha-1,4-linked glucose. We confirmed this by iodine staining with Lugol's iodine, which dyes glycogen in a brown color. If more glycogen is present, the color of stainend cultures is darker. In the picture below, the BL21 Gold (DE3) ∆glgP strain is much darker than the wild type.
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| glgP gene knockout The knockout was achieved cutting out most of the coding sequence of glgP, thereby inserting a short coding sequence that contains multiple stop codons in both frames. |
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| Iodine staining of BL21 Gold (DE3) glgP knockout compared to BL21 Gold (DE3) wild type Iodine staining was performed with overnight cultures in M9 medium which were adjusted to the same OD. The darker color of the glgP knockout strain shows that more glycogen is present. Thus, the effectivity of the targeting plasmid is shown. |
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 375
Illegal XbaI site found at 381
Illegal SpeI site found at 132
Illegal SpeI site found at 266
Illegal PstI site found at 393 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 375
Illegal NheI site found at 155
Illegal NheI site found at 243
Illegal SpeI site found at 132
Illegal SpeI site found at 266
Illegal PstI site found at 393 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 375
Illegal BglII site found at 1238
Illegal BamHI site found at 23 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 375
Illegal XbaI site found at 381
Illegal SpeI site found at 132
Illegal SpeI site found at 266
Illegal PstI site found at 393 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 375
Illegal XbaI site found at 381
Illegal SpeI site found at 132
Illegal SpeI site found at 266
Illegal PstI site found at 393
Illegal AgeI site found at 1088 - 1000COMPATIBLE WITH RFC[1000]
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