Composite

Part:BBa_K1632022:Design

Designed by: Jun kawamura   Group: iGEM15_Tokyo_Tech   (2015-08-30)
Revision as of 22:58, 13 September 2015 by JunKawamura (Talk | contribs) (→‎Materials and Methods)


J23100_rbs_lasR_TT_Plux_rbs_CmRssrA


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 383
    Illegal AgeI site found at 580
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 927


Design Notes

sequence confirmed

Materials and Methods

1.Construction
All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.

A.Pcon_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)
B.Pcon_lasR_TT_Plux_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)
C.Pcon_lasR_TT_⊿P_CmR (pSB6A1) + Plac_rhlI (pSB3K3)…Negative control #1
D.Pcon_lasR_TT_⊿P_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)…Negative control #2
E.Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) + Plac_rhlI (pSB3K3)
F.Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) + promoter less_rhlI (pSB3K3)

[[Image:|thumb|center|600px|Fig. 1. Plasmids]]

2. Assay protocol
1.Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2.Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.
3.Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4.Suspend the pellet in 1 mL of LB containing Amp and Kan.
5.Add 30 microL of suspension in the following medium.
　　a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (6 microL of 25 microg/mL) + 99.5% ethanol (6 microL)
　　b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (9 microL of 25 microg/mL) + 99.5% ethanol (3 microL)
　　c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (12 microL of 25 microg/mL)
6.Grow the samples of cells at 37°C for more than 8 hours.
7.Measure the optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)

Source

Composite of BBa_K553003, BBa_K1632021

References