Difference between revisions of "Part:BBa K1632023:Experience"
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A.Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)<br> | A.Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)<br> | ||
| − | B.Pcon_rhlR_TT_Plux_CmR (pSB6A1) + | + | B.Pcon_rhlR_TT_Plux_CmR (pSB6A1) +promotor_lasI (pSB3K3)<br> |
| − | C. | + | C.Pcon_rhlR_TT_promotor_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1<br> |
| − | D. | + | D.Pcon_rhlR_TT_promotor_CmR (pSB6A1) +promotor_lasI (pSB3K3)…Negative control #2<br> |
E.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)<br> | E.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)<br> | ||
| − | F.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + | + | F.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) +promotor_lasI (pSB3K3)<br> |
[[Image:RhlR cmRssrA Assay Construction.png|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br> | [[Image:RhlR cmRssrA Assay Construction.png|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br> | ||
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4.Suspend the pellet in 1mL of LB containing Amp and Kan.<br> | 4.Suspend the pellet in 1mL of LB containing Amp and Kan.<br> | ||
5.Add 30 microL of suspension in the following medium.<br> | 5.Add 30 microL of suspension in the following medium.<br> | ||
| − | a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 microL) + 99.5% ethanol (3 microL)<br> | + | <span style="margin-left: 20px;">a)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 microL) + 99.5% ethanol (3 microL)<br> |
| − | b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + 99.5% ethanol (3 microL)<br> | + | <span style="margin-left: 20px;">b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + 99.5% ethanol (3 microL)<br> |
| − | c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 microL) + Chloramphenicol (100 microg/mL)<br> | + | <span style="margin-left: 20px;">c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 microL) + Chloramphenicol (100 microg/mL)<br> |
| − | d)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (100 microg/mL)<br> | + | <span style="margin-left: 20px;">d)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (100 microg/mL)<br> |
6.Grow the samples of cells at 37°C for more than 8 hours.<br> | 6.Grow the samples of cells at 37°C for more than 8 hours.<br> | ||
7.Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)<br | 7.Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)<br | ||
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===Applications of BBa_K1632023=== | ===Applications of BBa_K1632023=== | ||
Revision as of 03:01, 16 September 2015
J23100_rbs_rhlR_TT_Plux_rbs_CmRssrA
Materials and Methods
1.Construction
All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.
A.Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)
B.Pcon_rhlR_TT_Plux_CmR (pSB6A1) +promotor_lasI (pSB3K3)
C.Pcon_rhlR_TT_promotor_CmR (pSB6A1) + Plac_lasI (pSB3K3)…Negative control #1
D.Pcon_rhlR_TT_promotor_CmR (pSB6A1) +promotor_lasI (pSB3K3)…Negative control #2
E.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)
F.Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) +promotor_lasI (pSB3K3)
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 antibiotic 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 1mL 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) + 50 microL C4HSL (30 microL) + 99.5% ethanol (3 microL)
b)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + 99.5% ethanol (3 microL)
c)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microL C4HSL (30 microL) + Chloramphenicol (100 microg/mL)
d)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (100 microg/mL)
6.Grow the samples of cells at 37°C for more than 8 hours.
7.Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)<br
Applications of BBa_K1632023
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