pGERM vector (Cat. No.: V017496)

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Basic Information

Note: pGERM is a suicide vector plasmid designed for gene disruption in Bacteroidesspecies and some fungi. It contains selectable markers for both E. coli(e.g., ampicillin resistance gene, bla) and the target organism (e.g., erythromycin resistance gene, ermG, for Bacteroides). It also includes an origin of transfer (oriTfrom RK2) for conjugation and a multiple cloning site. Since it cannot replicate in Bacteroides, selection for erythromycin resistance allows for the identification of clones where the plasmid has integrated into the chromosome via homologous recombination, enabling targeted gene disruption.

Name:: pGERM

Antibiotic Resistance:: Ampicillin

Length:: 4807 bp

Type:: Bacteroides Plasmid

Source/Author:: Shoemaker,N.B., Wang,G.R. and Salyers,A.A.

Copy Number:: High copy number

Growth Strain(s):: stbl3

Growth Temperature:: 37℃

$ 199.1

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

References

Hooper LV, Xu J, Falk PG, Midtvedt T, Gordon JI. A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem. Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9833-8. doi: 10.1073/pnas.96.17.9833. PMID: 10449780; PMCID: PMC22296.

pGERM vector (Cat. No.: V017496) Sequence

LOCUS       pGERM                   4807 bp    DNA     circular SYN 26-DEC-2025
DEFINITION  PGERM gene disruption vector, complete sequence.
ACCESSION   EF155418
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4807)
  AUTHORS   Shoemaker NB, Wang GR, Salyers AA.
  TITLE     Multiple gene products and sequences required for excision of the 
            mobilizable integrated Bacteroides element NBU1
  JOURNAL   J. Bacteriol. 182 (4), 928-936 (2000)
  PUBMED    10648516
REFERENCE   2  (bases 1 to 4807)
  AUTHORS   Chiang HC, Manchester JK, Gordon JI.
  TITLE     Regulation of nitrogen metabolism by a novel hybrid two-component 
            system protein in a prominent human gut symbiont, Bacteroides 
            thetaiotaomicron
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 4807)
  AUTHORS   Chiang HC, Manchester JK, Gordon JI.
  TITLE     Direct Submission
  JOURNAL   Submitted (03-DEC-2006) Center for Genome Sciences, Washington 
            University School of Medicine, 4444 Forest Park Ave. Campus Box 
            8510, Saint Louis, MO 63108, USA
REFERENCE   4  (bases 1 to 4807)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 4807)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J.
            Bacteriol."; date: "2000"; volume: "182"; issue: "4"; pages: 
            "928-936"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted
            (03-DEC-2006) Center for Genome Sciences, Washington University
            School of Medicine, 4444 Forest Park Ave. Campus Box 8510, Saint
            Louis, MO 63108, USA"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4807
                     /mol_type="other DNA"
                     /label=pGERM is pUC19 with the RK2 oriT and ermG
                     /note="pGERM is pUC19 with the RK2 oriT and ermG"
                     /db_xref="taxon:433620"
                     /organism="pGERM gene disruption vector"
     CDS             complement(44..367)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     277..293
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    294..350
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(363..379)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    387..403
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(411..441)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    456..477
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(709..1080)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            1113..1224
                     /label=origin of transfer from 56 kb circular plasmid RK2
                     /note="origin of transfer from 56 kb circular plasmid RK2"
     oriT            1113..1222
                     /label=incP origin of transfer
                     /note="incP origin of transfer"
     rep_origin      complement(1543..2131)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     gene            complement(2302..3162)
                     /gene="AmpR"
                     /label=AmpR
     CDS             complement(2302..3162)
                     /codon_start=1
                     /transl_table=11
                     /gene="AmpR"
                     /product="beta lactamase"
                     /label=AmpR
                     /protein_id="ABO69572.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHKMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            3650..4384
                     /gene="ermG"
                     /label=ermG
     CDS             3650..4384
                     /codon_start=1
                     /transl_table=11
                     /gene="ermG"
                     /product="rRNA methyltransferase"
                     /label=ermG
                     /protein_id="ABO69573.1"
                     /translation="MNKVNIKDSQNFITSKYHIEKIMNCISLDEKDNIFEIGAGKGHFT
                     AGLVKRCNFVTAIEIDSKLCEVTRNKLLNYPNYQIVNDDILKFTFPSHNPYKIFGSIPY
                     NISTNIIRKIVFESSATISYLIVEYGFAKMLLDTNRSLALLLMAEVDISILAKIPRYYF
                     HPKPKVDSTLIVLKRKPAKMAFKERKKYETFVMKWVNKEYEKLFTKNQFNKALKHARIY
                     DINNISFEQFVSLFNSYKIFNG"