Difference between revisions of "Part:BBa K4621079"

Latest revision as of 12:06, 11 October 2023

CRISPRi-62

This CRISPRi fragment contains the dCas9 and sgRNA necessary for the CRISPRi system. The CRISPRi system is suitable for a variety of Streptomyces.[1] In this study, it was used to inhibit the expression of GQS52 _ 08040 gene in SCUT-3 to produce more ectoine and hydroxyectoine.

Usage and Biology

Fig.1 CRISPRi system used in the study

Due to the limited types of plasmids available for SCUT-3, we inserted the CRISPRi-62 fragment into the previously constructed plasmid by homologous recombination to achieve simultaneous stable expression of multiple target genes. Subsequently, we verified the effectiveness of the CRISPRi-62 system (Pi2 in the figure below) in the fermentation of ordinary LB and shrimp shells.

Fig.2 Performance test on CRISPRi strains

Fig. 3 Ectoine/hydroxyectoine production using shrimp shell waste of CRISPRi strains

Reference

[1] Zhao Y, Li L, Zheng G, Jiang W, Deng Z, Wang Z, Lu Y. CRISPR/dCas9-Mediated Multiplex Gene Repression in Streptomyces. Biotechnol J. 2018 Sep;13(9):e1800121. doi: 10.1002/biot.201800121. Epub 2018 Jul 4. PMID: 29862648.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 15
Illegal SpeI site found at 4534
Illegal PstI site found at 3
Illegal PstI site found at 2428
Illegal PstI site found at 2662
Illegal PstI site found at 3874
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 4511
Illegal SpeI site found at 4534
Illegal PstI site found at 3
Illegal PstI site found at 2428
Illegal PstI site found at 2662
Illegal PstI site found at 3874
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 467
Illegal BglII site found at 1262
Illegal BglII site found at 4031
Illegal BamHI site found at 207
Illegal BamHI site found at 1556
Illegal BamHI site found at 4499
Illegal XhoI site found at 880
Illegal XhoI site found at 3226
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 15
Illegal SpeI site found at 4534
Illegal PstI site found at 3
Illegal PstI site found at 2428
Illegal PstI site found at 2662
Illegal PstI site found at 3874
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 15
Illegal SpeI site found at 4534
Illegal PstI site found at 3
Illegal PstI site found at 2428
Illegal PstI site found at 2662
Illegal PstI site found at 3874
Illegal NgoMIV site found at 1824
Illegal NgoMIV site found at 2195
Illegal NgoMIV site found at 2398
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 3406
Illegal BsaI.rc site found at 4285
Illegal SapI site found at 798
Illegal SapI site found at 2088
Illegal SapI.rc site found at 3285

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4621079 short</partinfo>
-This CRISPRi fragment contains four parts : BBa _ K4621070, BBa _ K3875019, BBa _ K4621029, and BBa _ K3081104, covering the dCas9 and sgRNA necessary for the CRISPRi system. The CRISPRi system is suitable for a variety of Streptomyces.[1] In this study, it was used to inhibit the expression of GQS52 _ 08040 gene in SCUT-3 to produce more ectoine and hydroxyectoine.
+This CRISPRi fragment contains the dCas9 and sgRNA necessary for the CRISPRi system. The CRISPRi system is suitable for a variety of Streptomyces.[1] In this study, it was used to inhibit the expression of GQS52 _ 08040 gene in SCUT-3 to produce more ectoine and hydroxyectoine.
 ===Usage and Biology===
@@ Line 19: / Line 19: @@
 https://static.igem.wiki/teams/4621/wiki/parts/fermentation-using-shrimp-shells-p.png
 Fig. 3 Ectoine/hydroxyectoine production using shrimp shell waste of CRISPRi strains
+===Reference===
+[1] Zhao Y, Li L, Zheng G, Jiang W, Deng Z, Wang Z, Lu Y. CRISPR/dCas9-Mediated Multiplex Gene Repression in Streptomyces. Biotechnol J. 2018 Sep;13(9):e1800121. doi: 10.1002/biot.201800121. Epub 2018 Jul 4. PMID: 29862648.
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