E. coli bacteria carrying RNAi plasmids that produce the desired dsRNA can be fed to worms, and the dsRNA is transferred to the worm via the intestinal tract. RNAi plasmids typically consist of DNA coding sequence from the intended target gene cloned between two T7lac promoters. The plasmid also has a selectable marker that confers resistance to an antibiotic, in this case ampicillin. In 7.15, you will use the E. coli strain HT115 carrying various L4440 plasmids, each containing a different cloned gene sequence. HT115 is an RNase III-deficient E. coli strain with IPTG-inducible T7 polymerase activity. To induce dsRNA production from these plasmids, the HT115 bacteria is grown on special RNAi NGM feeding plates that contain IPTG and the ampicillin analog carbenicillin. Carbenicillin is preferred over ampicillin because it tends to be more stable.
| 载体基本信息 | |||
| 载体名称 | L4440 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 2790 bp | 载体类型 | RNAi |
| 载体来源 | Andrew Fire | 拷贝数 | High copy number |
| 启动子 | T7 | 克隆方法 | Enzyme Cut |
| 表达方法 | IPTG induced | ||
L4440 载体载体图谱
L4440 载体载体序列
LOCUS Exported 2790 bp ds-DNA circular SYN 23-SEP-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS L4440
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 2790)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..2790
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 19..37
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind 95..111
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(181..197)
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(223..241)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(251..267)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 409..864
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 890..994
/gene="bla"
/note="AmpR promoter"
CDS 995..1855
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 2026..2614
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
