Team:Warsaw/Calendar-Main/5 August 2008

From 2008.igem.org

(Difference between revisions)
Line 51: Line 51:
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/e/ea/Western1_WAW.jpg" width=400/></a>
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/e/ea/Western1_WAW.jpg" width=400/></a>
-
<var><b>Fig. 1.</b>Appropriate description is needed!<Tutaj wstawię opis jak się wyśpię i będę zdolna do myślenia...></var>
+
<var><b>Fig. 1.Protein A detection in bacteria lysates.</b><br>
 +
1 and 2 - <i>E coli</i> without plasmid,<br>
 +
3 - Omp_omega_A_alpha + 0,5 mM IPTG,<br>
 +
4 - <i>E coli</i> without plasmid,<br>
 +
5 - Omp_omega_A_alpha + 0,5 mM IPTG,<br>
 +
6 - Omp_omega_A_alpha without inducer.</var>
<br><br>
<br><br>

Revision as of 22:24, 26 October 2008

Gallery Bricks Notebook Team Project Home


Previous day
return to main notebook page
Previous entry
next notebook entry

 



Cloning of truncated fragment of protein A (ΔA)

Emilia

  1. Isolation of plasmids from cultures inoculated on previous day.
  2. Control digest with SacI and BamHI (BamHI buffer).
  3. Gel electrophoresis - proper clones found for both products of ligation.

Checking if OmpA_omega_ΔA_alpha gives ampicillin resistance

Piotr

Inoculation OmpA_omega_ΔA_alpha from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.

Measurement of bacterial culture growth (OD) in the evening:

ampicillin concentration (μg/mL):IPTG concentration (mmol/mL):
00.10.250.50.751
251.5581.4691.5871.491.5661.311
501.4251.4351.5241.0550.9200.935
751.090.9891.4470.9710.9510.992
1000.090.6851.3781.0780.9770.992

Inoculation of OmpA_omega_ΔA_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (repetition is necessary)

Checking OmpA_omega_ΔA_alpha and OmpA_A_alpha expression

Piotr

  1. Spinning.
  2. Suspending.
  3. Adding of lysis buffer.
  4. Boiling.
  5. Putting into poliacrylamide gel.
  6. Transfer onto nitrocellulose.
  7. Blocking.
  8. Anti-A antibody binding.
  9. Washing.
  10. Anti-rabbit antibody binding.
  11. Developing with BCIP and NBT (Fig. 1.).
Fig. 1.Protein A detection in bacteria lysates.
1 and 2 - E coli without plasmid,
3 - Omp_omega_A_alpha + 0,5 mM IPTG,
4 - E coli without plasmid,
5 - Omp_omega_A_alpha + 0,5 mM IPTG,
6 - Omp_omega_A_alpha without inducer.


Preparing pACYC177+OmpA_omega_ΔA construct

Michał K.

  1. Transformation of E. coli TOP10 strain with ligation.
  2. Transformants plating on LB + kanamycin.