Team:UNICAMP-Brazil/Notebooks/October 15

From 2009.igem.org

(Difference between revisions)
(→Testing the new device)
(→Testing the new device)
 
(One intermediate revision not shown)
Line 15: Line 15:
==== Testing the new device ====
==== Testing the new device ====
-
*<p style=”text-align:justify;”>We confirmed our device yesterday, but we continued working with it.  We proved that our device really works. To fulfill this aim, we transformed competent ''E. Coli'' C43. This ''E. coli'' strain can produce the T7 polimeraze by IPTG induction.  Thus, when we put IPTG into the culture medium, we will have expression of T7 polimeraze, which will induce the T7 promoter of our device and then, the endolysin will be expressed. Our test consists in overexpression of the endolysin in order to break it's own peptidoglycan wall. We transformed cells with our device BBa_K284022 plasmid and with the BBa_K112806 plasmid (without promoter).</p>
+
*<p style=”text-align:justify;”> After we confirmed the construction of our device we start planning our characterization tests to prove that our device really works. To fulfill this aim, our new device was transformed in ''E. coli'' C43 strain, an overexpression strain in which the T7 promoter can be induced by IPTG. We transformed cells with our device, BBa_K284022, and with the BBa_K112806(without promoter).</p>
-
*<p style=”text-align:justify;”>So, after get the transformed cells, we put them to growth in 5 erlenmeyers (LB-AMP medium) until half log phase (OD-0.6 to 1). Then, we induced with IPTG (1mM) and measured the optical density during 4 hours after induction. We plated the 5 cultures in LB-AMP medium plates in order to confirm the diminished cellular growth.  To have a definitive confirmation, we also performed SDS-PAGE to notice the protein overexpression. The results are shown in [https://2009.igem.org/Team:UNICAMP-Brazil/Coliguard/Results ColiGuard results].</p>
+
*<p style=”text-align:justify;”>A transformed colony was incubated in LB medium with ampicillin at 37ºC overnight. The culture was diluted in new LB medium with ampicillin to an OD=0,2 and incubated at 37ºC until reach an OD=0,8. At this point, the cells were induced with IPTG 1 mM and incubated again for 4 hours. After the end of the induction period the cells were plated in LB agar with ampicillin in order to confirm the diminished cellular growth.  To have a definitive confirmation, we also performed SDS-PAGE to notice the protein overexpression. The results are shown in [https://2009.igem.org/Team:UNICAMP-Brazil/Coliguard/Results ColiGuard results].</p>
''Luige, Ane and Marcos''
''Luige, Ane and Marcos''

Latest revision as of 03:21, 22 October 2009

Topo l2.gif topo_r_igem.gif
topo_r_b.gif
Back to Calendar

ColiGuard

PY Promoter - Preparation of electrocompetent cells

  • Today we prepared electrocompetent cells of the conjugative strain following

Protocol 4.

Fabi and Léo

Testing the new device

  • After we confirmed the construction of our device we start planning our characterization tests to prove that our device really works. To fulfill this aim, our new device was transformed in E. coli C43 strain, an overexpression strain in which the T7 promoter can be induced by IPTG. We transformed cells with our device, BBa_K284022, and with the BBa_K112806(without promoter).

  • A transformed colony was incubated in LB medium with ampicillin at 37ºC overnight. The culture was diluted in new LB medium with ampicillin to an OD=0,2 and incubated at 37ºC until reach an OD=0,8. At this point, the cells were induced with IPTG 1 mM and incubated again for 4 hours. After the end of the induction period the cells were plated in LB agar with ampicillin in order to confirm the diminished cellular growth. To have a definitive confirmation, we also performed SDS-PAGE to notice the protein overexpression. The results are shown in ColiGuard results.

Luige, Ane and Marcos

YeastGuard

New Strategy: pGEM

  • We sent 4 biobricks to iGEM today: pJEN1, pDLD, Lysozyme, and the devices Adh1+YFP and Adh1+Lysozyme. =)

Yeast experiments

  • We transformed the YEP+Adh1-lysozyme ligation reaction in competent E. coli and plated in LB+Amp media. Hope to have colonies later!

  • We did miniprep of YEP+Adh1-YFP and the following devices: b) pJEN1+YFP, c) pJEN1+Lys, d) pDLD+YFP, e) pDLD+Lys.

  • We did the pre inoculun to prepare competet yeast tomorrow.


Raíssa and Taís




Retrieved from "http://2009.igem.org/Team:UNICAMP-Brazil/Notebooks/October_15"