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.


    Protocol:

    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


    ACCESSION   .


    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 


                         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="pUC ori"


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


                         replication"


    ORIGIN


            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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    Product is for research use only!