L4440 nematode plasmid

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    L4440 Information

    Use:Nematode plasmid

    Promoter: T7

    Replicator: pUC ori, F1 ori

    Plasmid classification: nematode RNAI interference vector; Worm Expression RNAi

    Plasmid size: 2790bp

    Plasmid label: lacZN, OriF1

    Prokaryotic resistance: ampicillin Ampicillin

    Cloned strain: DH5 alpha

    Culture conditions: 37 centigrade, aerobic, LB

    Expression host: nematode, worm

    Induction mode: IPTG

    5'sequencing primers: Based on full sequence design

    L4440 Description:

    Reverse Genetics and RNAi:

    RNAi is a natural cellular process in many eukaryotes which scientists have taken advantage of in the lab as a valuable reverse genetics mechanism for regulating gene expression. RNAi involves double-stranded RNA (dsRNA) interfering with the expression of genes with sequences complementary to the dsRNA. By administering a specific dsRNA to a cell culture or multicellular organism, RNAi can be induced to selectively reduce expression of that specific gene in the cells or organism.

    RNAi has been  particularly well studied in  C. elegans, in which the RNAi gene silencing phenotype is heritable and the dsRNA is easily administered.  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.


    A. Preparing feeding plates 

    1) Inocluate 3mL of LB containing 50 µg/mL  ampicillin with individual desired bacterial strain. Grow overnight at 37oC.

    2) Seed NGM agar feeding plates  (containing 25 µg/mL  carbenicillin  and 1mM IPTG) by pipetting 150 µL of LB bacterial culture into the center of the plate.  You should have three RNAi feeding plates:

    a. One plate will be seeded with HT115 bacteria carrying the “empty” L4440 vector (no C. elegans gene is cloned in the vector).

    b. Another plate will be seeded with HT115 bacteria carrying a portion of the unc-22 gene cloned into the L4440 vector. The phenotype resulting from RNAi of unc-22 is well-established and is reliably reproducible under the experimental conditions you are using today.

    c. A third plate will be seeded with HT115 bacteria carrying a portion of your gene of interest cloned into the L4440 vector.  We will use dpy10 as our gene of interest.

    3) Let the plates dry overnight at room temperature.

    B. Transfer N2 L4 worms 

    1) Transfer two L4 hermaphrodites  from the N2  stock plate to each of the RNAi  feeding plates, minimizing the amount of OP50 bacteria transferred. Try to avoid bringing any younger contaminating worms along with the L4 worms you are transferring.

    2) Incubate the plates until the progeny reach the desired age (Note: it takes ~4 days for a C. elegans egg to grow into a gravid adult when grown at 16oC.

    C. Scoring RNAi phenotypes

    1) Observe RNAi controls. Start by looking at the L4440 RNAi plates  - what phenotype(s) would you expect to see on these plates? Next, look at the unc-22 RNAi plates – what phenotype(s) would you expect to see on these plates? Record all your observations in your notebook. If you do not see the expected phenotypes on your control plates, this may indicate your RNAi experiment was set-up incorrectly.

    2) Observe the RNAi phenotypes  for the experimental RNAi construct and record all your observations in your notebook. Examine  how the observed phenotypes  compare with the corresponding null mutant phenotypes  in the same gene (the WormBase database has information on previously characterized RNAi and null mutant phenotypes).

    L4440 Sequence:

    LOCUS       Exported                2790 bp ds-DNA     circular SYN 04-SEP-2016

    DEFINITION  synthetic circular DNA


    VERSION     .

    KEYWORDS    L4440

    SOURCE      synthetic DNA construct

      ORGANISM  synthetic DNA construct

    REFERENCE   1  (bases 1 to 2790)

      AUTHORS   .

      TITLE     Direct Submission

      JOURNAL   Exported Sunday, September 4, 2016 from SnapGene Viewer 3.1.4

    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 


         primer_bind     complement(181..197)

                         /note="KS primer"

                         /note="common sequencing primer, one of multiple similar 


         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 


         rep_origin      409..864


                         /note="f1 ori"

                         /note="f1 bacteriophage origin of replication; arrow 

                         indicates direction of (+) strand synthesis"

         promoter        890..994


                         /note="AmpR promoter"

         CDS             995..1855





                         /note="confers resistance to ampicillin, carbenicillin, and

                         related antibiotics"







         rep_origin      2026..2614


                         /note="pUC ori"

                         /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 



            1 aacctggctt atcgaaatta atacgactca ctatagggag accggcagat ctgatatcat

           61 cgatgaattc gagctccacc gcggtggcgg ccgctctaga actagtggat ccaccggttc

          121 catggctagc cacgtgacgc gtggatcccc cgggctgcag gaattcgata tcaagcttat

          181 cgataccgtc gacctcgagg gggggcccgg tacccaattc gccctatagt gagtcgtatt

          241 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc

          301 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc

          361 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta

          421 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca

          481 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct

          541 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc

          601 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat

          661 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc

          721 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc

          781 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta

          841 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc

          901 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg

          961 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc

         1021 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt

         1081 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct

         1141 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac

         1201 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact

         1261 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa

         1321 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga

         1381 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt

         1441 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga

         1501 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg

         1561 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat

         1621 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat

         1681 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc

         1741 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga

         1801 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc

         1861 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag

         1921 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc

         1981 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt

         2041 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt

         2101 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat

         2161 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc

         2221 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa

         2281 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg

         2341 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag

         2401 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag

         2461 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa

         2521 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt

         2581 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg

         2641 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc

         2701 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac

         2761 cgagcgcagc gagtcagtga gcgaggaagc



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