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HT115(DE3) strain-BioVector NTCC保藏中心

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HT115(DE3)菌株

Species:

Escherichia coli


HT115 (DE3) genotype


F-, mcrA, mcrB, IN(rrnD-rrnE)1,lambda -, rnc14::Tn10(DE3 lysogen: lavUV5 promoter -T7 polymerase)(IPTG-inducible T7 polymerase) (RNase III minus).



Bacteria-mediated RNAi--General outline:

Lisa Timmons

Carnegie Institution of Washington

Updated May 2000

1) Place gene of interest between T7 promoters in “double T7” plamsid. (slightly more difficult alternative: Place gene of

interest in hairpin/inverted repeat configuration behind T7 promoter in pBlueScript.)

note: this (and other standard cloning) should be performed in DH5 bacteria or other standard cloning strain--NOT

HT115(DE3) cells.

another note: The double T7 promoter-containing plasmid as well as control plasmids for use in feeding experiments

are availale in the 1999 FireLab vector kit. Information and kit request forms can be accessed through the Carnegie

Web site: http://www.ciwemb.edu/

2) Transform plasmid into competent HT115(DE3) bacterial cells and plate onto standard LB+ tetracycline+antibiotic plates.

(Easy competent cell and transformation protocols below)

note: the HT115 cells were a generous gift from D. Court (NCI).

another note: the HT115(DE3) strain is tetracycline resistant; nonetheless, care must be taken not to contaminate your

bacterial stock. First plate the cells onto TET plates, immediately freeze an aliquot upon receipt, and also freeze any

transformed strains in order to have reliable backup. (see freezing protocol below). In addition, the only reliable way

to verify the presence of the DE3 lysogen is by PCR, since T7 phage will not grow in the RNAseIII- background of

this cell.

yet another note: The HT115(DE3) strain is now available from the CGC. (http://biosci.cbs.umn.edu/CGC/

CGChomepage.htm)

3) Grow up culture from single colony on plates and induce expression of dsRNA using IPTG (induction protocol below).

4) Seed NGM plates with the induced culture. The culture can be used as is (for small plates containing small numbers of

hand-picked worms, eg), or the cells can be concentrated by centrifugation and spotted onto plates (for large plates containing

chunked worms, eg). The ratio of bacteria to worms is important--If the plates starve out, RNAi will not be effective. In

addition, the bacterial lawn should not be allowed to continue to grow. Cells that do grow on plates after induction are

generally cells that have lost the plasmid, cells that have lost the ability to produce T7 polymerase, or cells that are

contaminants. The inclusion of tetracycline in the plates significantly improves the results (the addition of ampicillin also helps

—in the case of amp resistant plasmids) (50ug/ml AMP,12.5ug/ml TET). IPTG included in the plates does not significantly

improve the RNAi phenotypes in my hands, but I usually include it in the NGM plates anyway, especially if the seeded plates

are not going to be used immediately (0.4mM IPTG).

5) Add worms to plate and incubate at appropriate temperature. Worms can be added by hand-picking or by adding chunks

onto wet, freshly seeded plates, or onto plates that have been allowed to dry after seeding. I generally use freshly seeded plates

in my experiments. Older seeded plates containing IPTG can also induce RNAi phenotypes with good success. We observe

phenotypes at all temperatures from 16-25C, although the expressivity and penetrance of the phenotype can vary depending

upon the incubation temperature and gene. Worms grown on dsgfp-plates have a stronger RNAi phenotypes when the plates

are incubated at lower temps (16C or 20C), with ds unc-22, phenotypes more convincing at higher temps (25C). It can take

three days before an RNAi phenotype is observed. Results vary depending on the dsRNA and the worm strains used. Freshly

seeded plates vs older seeded plates is also a consideration.

Quick procedure for making competent bacterial cells using CaCl2:

1) Inoculate overnight culture in LB + antibiotic (TET for HT115(DE3) strain + antibiotic appropriate for any plasmids in

cells) (2-5ml). Shake overnight at 37C.2) Inoculate 25 ml LB + antibiotic with overnight culture, 1:100 dilution. Grow cells to OD595= 0.4. Can grow cells in 50

ml sterile centrifuge tube.

3) Spin cells 10 min 3000 rpm at 4C.

4) Resuspend pellet in 0.5X original volume cold, sterile 50 mM CaCl2 (12.5ml). Resuspend by GENTLY pipetting up and

down a few times with a wide bore pipet--no vortexing.

5) Incubate on ice 30 min.

6) Spin as before at 4C.

7) Resuspend pellet as before in 0.1X original volume CaCl2 (2.5ml). Keep cells cold (4C).

8) Use 50-200 ul for transformation.

Cells can be used as is for up to three days (stored at 4C). The cells can be frozen by: adding glycerol to final concentration

of 10%, rapid freezing on dry ice/etoh, and storing at –80.

Transformation of CaCl2 competent HT115(DE3):

1) Add 50-200 ul competent cells to cold, sterile, polypropylene tube on ice.

2) Add 1 uL plasmid (mini-prep quality is OK) 1-100ng.

3) Incubate on ice/water bath for 30 min.

4) Immerse tube in 37C water bath for 1 min.

5) Incubate tube on ice/water bath for 2 min.

6) Add 1ml sterile SOC media. Incubate 37C with shaking for 1 hour.

7) Plate 10uL, 100uL, 250uL, and remaining culture onto 4 LB + tetracycline + other antibiotic? (plasmid resistance) plates.

Incubate 37C overnight. (These cells grow slowly, allow 36 hours for colony formation.)

Freezing bacterial stocks:

1) Inoculate fresh single colony of bacteria into 2.5 ml LB+ antibiotic(s). Grow to early stationary phase.

2) Pipette 0.25 ml 80% glycerol (sterile) and 0.75 ml culture into a sterile screw-cap freezer tube. Mix.

3) Quick freeze on dry ice/ethanol and store at -80C.

Induction of dsRNA in HT115(DE3) cells + T7 promoter containing plasmid:

1) Inoculate overnight culture of HT115(DE3) + plasmid in LB+antibiotics. Incubate 37C with shaking overnight.

(75-100ug/ml ampicillin for amp-resistant plasmids and 12.5 ug/ml tetracycline.)

2) Dilute culture 1:100 in 2xYT + antibiotics and grow to OD595=0.4. (A 25ml culture is usually enough for a small

experiment ~ 20 small plates.)

3) Induce by adding sterile IPTG to 0.4 mM. Incubate 37C with shaking ~ 4 hours (see note).

4) Spike culture with additional antibiotics (another 100ug/ml AMP and 12.5ug/ml TET) and IPTG (to final total concentration

of 0.8mM). Seed plates using culture as is or concentrate cells by centrifugation as per general protocol.

note: the same parameters which give variable results in protein expression using the T7 promoter system are also variable in

this system. Variables to consider: induction temperature (37C vs 30C), induction time (2hr, 4hr, overnight--although

overnight induction never works for me), concentration of IPTG, induction volume, media (LB vs 2xYT or other medias),

additives to induction media (uracil, lactose,etc), etc. Alterations from this "standard" protocol have not significantly or

reproducably improved the RNAi results for the genes we have tested. In addition, “fresh” cells tend to work best. Bacteria

that have been stored on plates at 4C for a long period of time often lose effectiveness: try streaking a new plate from frozen

cells+plasmid or try retransforming HT115(DE3) with the plasmid of interest.

Bacterial strains

Bacterial StrainStrain DescriptionDetails
HT115(DE3)HT115(DE3) E. coliRNAi feeding strain
HT115(DE3)/pL4400Empty RNA1 feeding vector L4440 in feeding strainAmpicillin selection No phenotype
HT115(DE3)/pL4400(bli-1)blister-1 RNAi feeding straininduces blister by RNAi
HT115(DE3)/pL4400(dpy-10)dpy-10 RNAi feeding straininduces dumpy by RNAi
HT115(DE3)/pL4400(rol-5)rol-5 RNAi feeding straininduces roller phenoytpe by RNAi
HT115(DE3)/pL4400(T04A11.6)him-6 RNAi feeding straininduces high incidence of males by RNAi
HT115(DE3)/pL4400(unc-22)unc-22 RNAi feeding straininduces twitcher by RNAi
HT115(DE3)/pL4400(unc-23)unc-23 RNAi feeding straininduces uncoordination by RNAi
OP50Uracil auxotroph E. coli B.feeding behavior for C. elegans
AttachmentSize
c.elegansfeedinglibrary-individualclones.pdf87.23 KB

Competent cells

Protocol for making competent cells

Need competent cells for students in week 3 - make them in week 1 or 2.

0.5 M PIPES disodium salt(pH 6.7) (Alfa Aesar 3p B21835-22) or Fisher AC21509-1000

  • 17.3 g PIPES (disodium salt) in 80 mL H2O

  • OR 15.1 g PIPES in 80 mL H2O

  • initial pH = ~5.5

  • KOH pellets till it goes clear (checking pH!!)

  • final pH to 6.7 with 5M KOH

  • water to 100 mL

  • filter sterilize

  • freeze in 10 mL aliquots (enough for 500 mL transformation buffer)

Transformation buffer

Reagentcf (mM)per L.
MnCl25510.88g
CaCl2152.2g
KCl25018.65g
PIPES (pH 6.7, 0.5M)1020mL

filter sterilize. refrigerate


SOB Medium

Per Liter:

  • 950 ml of deionized H2O

    • 20 g Tryptone

    • 5 g Yeast Extract

    • 0.5 g NaCl


  • stir to dissolve

    • 2.5 mL 1M KCl (10 mL 250 mM KCl)


  • pH to 7 with NaOH

  • volume to 1 L

  • autoclave 20 min

  • cool

    • 10 mL sterile 1M MgCl (5 mL sterile MgCl2 2M)

    • ? 10 mL sterile 1M MgSO4?


SOB AGAR

15 g agar/L SOB

when cooled, add

20 mL 1 M MgSO4

12.5 µg/mL tetracycline (=0.83 mL 15 mg/mL tet per L)

or

50 µg/mL ampicillin as needed (= 1mL 50 mg/mL amp stock per L)

LB amp

LB ampicilin plates for transformation

48 plates x 25 mL/plate = 1200 mL

Make 1500 mL:

25 g LB + 15 g agar /liter and 50 µg/mL ampicillin

http://wahoo.nsm.umass.edu/content/ampicillin

37.5 g LB in 1500 mL water in a 2L flask

stir to dissolve, leave stir bar in.

add 22.5 g agar

Autoclave 50 min. Include PourBoy tubing if necessary, and 2 flasks of water.

Cool till handle-able

Add 1500 µL 50 mg/mL amp

Stir

Dispense 25 mL/plate

NGM plates

Per Liter of medium (~75 plates):

  • 975 mL Water

  • 3 g NaCl

  • 2.5 g Peptone (Fisher BP1420-500 $78.80)

  • 17 g Bactoagar

This is what I did by mistake: per 1.5 L (112 plates):

  • 1462 mL Water

  • 18 g NaCl

  • 15 g Peptone (Fisher BP1420-500 $78.80)

  • 25.5 g Bactoagar (haven't added it yet)

So I labeled it NGM 4X.

I made 4 liters by diluting 975 mL 4xNGM with 975 x 3 mL water, then added 68 g bactoagar.

Stir flask to distribute ingredients.

Autoclave with stir bar inside

Cool to 55C in a 55C water bath

Add per L (see recipes in stock solutions):

  • 1 mL cholesterol (5 mg/mL in 95% EtOH)

  • 1 mL CaCl2 (1 M, STERILE)

  • 1 mL MgSO4 (1 M, STERILE)

  • 25 mL K-phosphate buffer (1M, pH 6.0, STERILE1)

Swirl flask to mix

Dispense 10 mL into each 60mm dish.

Stack 10 high

Let stand for ~48 hours for condensation to evaporate

Pack in sterilized plastic boxes.


  1. 3.3 mL K2HPO4 + 21.7 mL KH2PO4 ↩

OP broth

B Broth (for OP50 E. coli to feed to worms)

  • 10 g bactotryptone

  • 5 g NaCl

  • 1 L dH2O

Autoclave

400 µL overnight culture to seed plates.

Store for months in refrigerator.

200 mL cells ON for 500 plates

(can save the extra ON culture in the refrigerator for 3 days.)

From WormBase:

3.1. Preparation of bacterial food source

Although C. elegans can be maintained axenically (Avery, 1993), it is difficult, and the animals grow very slowly. C. elegans is usually grown monoxenically in the laboratory using E. coli strain OP50 as a food source (Brenner, 1974). E. coli OP50 is a uracil auxotroph whose growth is limited on NGM plates. A limited bacterial lawn is desirable because it allows for easier observation and better mating of the worms. A starter culture of E. coli OP50 can be obtained from the CGC or can be recovered from worm plates. Use the starter culture to isolate single colonies on a streak plate of a rich medium such as LB agar [10 g Bacto-tryptone, 5 g Bacto-yeast, 5 g NaCL, 15 g agar, H2 O to 1 litre, pH 7.5] (Byerly et al., 1976). Using a single colony from the streak plate, aseptically inoculate a rich broth, such as L Broth [10 g Bacto-tryptone, 5 g Bacto-yeast, 5 g NaCl, H2 O to 1 litre, pH to 7.0 using 1 M NaOH. Put 100 ml into 250 ml screw-cap bottles and autoclave. The bottles of media can be stored at room temperature for several months (Byerly et al., 1976)]. Allow inoculated cultures to grow overnight at 37°C. The E. coli OP50 solution is then ready for use in seeding NGM plates. The E. coli OP50 streak plate and liquid culture should be stored at 4°C and will remain usable for several months.


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