Difference between revisions of "Part:BBa K1632011:Design"

(→‎Assay protocol)
Line 31: Line 31:
 
=====Assay protocol=====
 
=====Assay protocol=====
  
−
1. Prepare overnight cultures for the each sample in 3 mL of LB medium containing ampicillin (50 microg / mL), kanamycin (30 microg / mL)　and glucose (final concentration of mass of glucose is 1.0 %) at 37 ℃ for 12h.<br>
+
1. Prepare overnight cultures for each sample in 3 mL of LB medium containing ampicillin (50 microg / mL), kanamycin (30 microg / mL)　and glucose (final concentration is 1.0 %) at 37 ℃ for 12h.<br>
−
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration of mass of glucose is 1.0 %).<br>
+
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration is 1.0 %).<br>
−
3.Grow the cells at 37 ℃, shaking at 180 rpm until the observed OD590 reaches 0.4 (Fresh Culture).<br>
+
3. Incubate the cells at 37 ℃, shaking at 180 rpm until the observed OD590 reaches 0.4 (Fresh Culture).<br>
−
4. After incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
+
4. After the incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 
5. Remove the supernatant.<br>
 
5. Remove the supernatant.<br>
 
6. Add 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 
6. Add 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
Line 42: Line 42:
 
10. Suspend the pellet in 1mL of LB containing Amp and Kan.<br>
 
10. Suspend the pellet in 1mL of LB containing Amp and Kan.<br>
 
11. Add 30 microL of suspension in the following medium.<br>
 
11. Add 30 microL of suspension in the following medium.<br>
−
<span style="margin-left: 20px;">① 3 mL of LB containing Amp, Kan and 30 microL of sterile water<br>
+
<span style="margin-left: 20px;">① 3 mL of LB containing Amp, Kan, glucose (final concentration of glucose is 1.0 %) and 30 microL sterile water<br>
 
<span style="margin-left: 20px;">② 3 mL of LB containing Amp, Kan and 30 microL of 500 microM　arabinose (final concentration of arabinose is 5 microM)<br>
 
<span style="margin-left: 20px;">② 3 mL of LB containing Amp, Kan and 30 microL of 500 microM　arabinose (final concentration of arabinose is 5 microM)<br>
 
<span style="margin-left: 20px;">③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 10 microM)<br>
 
<span style="margin-left: 20px;">③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 10 microM)<br>

Revision as of 02:00, 18 September 2015

fimE (wild-type)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 22
  • 1000
    COMPATIBLE WITH RFC[1000]



2008_Caltech FimE(BBa_K137007) didn’t match the nucleotide sequence of fimE of wild type. So, we designed fimE(wild-type) (BBa_K1632011) which completely match the nucleotide sequence of fimE of wild type. Compared with these two parts, two differences were confirmed. First, fimE of 2008_Caltech lacks the nucleotide sequence of N-terminal 15 residues. Second, fimE of 2008_Caltech is different from the nucleotide sequence of C-terminal 2 residues as shown as below.
2008_Caltech : 5’-...-TAA-TAA-Suffix-3’
2015_TokyoTech : 5’-...-GTT-TGA-Suffix-3’


Design Notes

sequence confirmed

Materials and Methods

Construction

All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.

A. PBAD/araC_fimE(wild-type) (pSB6A1) + fim switch[default ON](wild-type)_rbs_gfp (pSB3K3)
B. PBAD/araC_fimE(wild-type) (pSB6A1) + fim switch[default OFF](wild-type)_rbs_gfp (pSB3K3)
C. pSB6A1 + fim switch[default ON](wild-type)_rbs_gfp (pSB3K3) …positive control 1
D. pSB6A1 + fim switch[default OFF](wild-type)_rbs_gfp (pSB3K3) …negative control 1
E. PBAD/araC_fimE(wild-type) (pSB6A1) + J23119_rbs_gfp (pSB3K3) …positive control 2
F. PBAD/araC_fimE(wild-type) (pSB6A1) + rbs_gfp (pSB3K3) …negative control 2

Fig. 1. Plasmids

Assay protocol

1. Prepare overnight cultures for each sample in 3 mL of LB medium containing ampicillin (50 microg / mL), kanamycin (30 microg / mL)　and glucose (final concentration is 1.0 %) at 37 ℃ for 12h.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration is 1.0 %).
3. Incubate the cells at 37 ℃, shaking at 180 rpm until the observed OD590 reaches 0.4 (Fresh Culture).
4. After the incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃
5. Remove the supernatant.
6. Add 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃
7. Remove the supernatant.
8. Take the samples, and centrifuge at 5000x g, 1 min, 25 ℃
9. Remove the supernatant.
10. Suspend the pellet in 1mL of LB containing Amp and Kan.
11. Add 30 microL of suspension in the following medium.
① 3 mL of LB containing Amp, Kan, glucose (final concentration of glucose is 1.0 %) and 30 microL sterile water
② 3 mL of LB containing Amp, Kan and 30 microL of 500 microM　arabinose (final concentration of arabinose is 5 microM)
③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 10 microM)
④ 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)
※ As for C and D, the suspension were added only in medium ① and ④.
12. Incubate the samples at 37 ℃ for 6 hours, shaking at 180 rpm. (Measure the OD590 of all the samples every hour.)
13. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.
14. Remove the supernatant.
15. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)
16. Dispense all of each suspension into a disposable tube through a cell strainer.
17. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)

Source

PCR from MG1655

===References===