Team:UC Chile/Cyano/Labook/september

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Revision as of 21:41, 12 October 2012

Project: Luxilla - Pontificia Universidad Católica de Chile, iGEM 2012

September

BBa¬_K743015 assembled during the last days of August was verified by sequencing.

Also during the last days of August, Synechocystis PCC 6803 was transformed with:

Int_C BBa_K743004 BBa_K743014 BBa_K743016 psbAB+GFP+pPMQAK1

Only Int_C could not be transformed into cells. A colony PCR was run to amplify different sequences of BBa_K743004 and psbAB+GFP+pPMQAK1. Positive results for only for para BBa_K743004.

BBa_K743014 was inoculated in a 500 mL flask to test induction by decanal. The inoculum grew in Kanamycin but did not produce bioluminescence when induced by decanal.

BBa_K743004 and psbAB+GFP+pPMQAK1 was replated in Kanamycin plates but there was no growth. Construct psbAB+GFP+pPMQAK1 was replated in Kanamycin three times, but never grew. We believe it could not be transformed.

We transformed Synechosystis pcc 6803 again with the following parts and now cells are being incubated at optimal conditions (continuous light and 30 °C).

BBa_K743004 BBa_K743009 BBa_K743016 BBa_K743006 BBa_K743014 BBa_K743015 IntC Control – (KAN) Control – (CLO)

IntC could not be transformed again. The rest of the transformations seem ok.

BBa_K743014 and BBa_K743016 were plated in kanamycin plates from transformation plates and a colony PCR was run for BBa_K743014 amplifying LuxAB and LuxAB with promoter.

A new round of Synechocystis was run under the same conditions and constructs that the last one.

The last two transformations resulted in negative controls and individual colonies for most constrcuts except for IntC (did not transform). Colonies were picked from all transformations and were replated in kanamycin plates. Our cells grew in kanamycin, which indicates they are transformed. Also, the plates were inoculated I nliquid media with kanamycin and they also grew.

After multiple replatings in Kanamycin, a massive colony PCR was run of the transformed cells from the last two rounds of transformation. BBa_K743015 and BBa_K743016 transformations were verified.

Due to the important amount of failed attempts to transform IntC plasmid from Utah, this was digested resulting in bands of wrong size. We sent it to sequencing.

Characterization of LuxBrick was done.

A growth curve was characterized for Synechosystis PCC 6803 from an OD730:0.4800 inoculum.

Inoculums: 3 flasks with 1 mL in 150 mL of normal BG11. 1 control with 1 mL in 150 mL of BG110.

Shaker at 90 RPM.

Experiment for characterization of Gibson for small parts was run.

We tried to see LuxBrick induced under microscope. We concluded bioluminescence can’t be seen under microscope.

We amplified parts and run a Gibson to assemble BBa_K743017 and BBa_K743018. Transformation and verification by digestion and PCR was done afterwards. Constructs were sent to sequence.

Biolumiscence of different constructs with luciferase was measured when induced with different aldehides (decanal and dodecanal) using an Illumeter.

@@ Line 39: / Line 39: @@
 <p><font size= "5">September</font></p>
-(in spanish)
-RESUMEN SEPTIEMBRE
+BBa¬_K743015 assembled during the last days of August was verified by sequencing.
-Se mando a secuenciar y validó BBa_K743015 obtenido los últimos días de agosto.
+Also during the last days of August, Synechocystis PCC 6803 was transformed with:
-A finales de agosto se hizo una transformación de Synechosystis pcc 6803 con:
 Int_C
 BBa_K743004
 BBa_K743014
 BBa_K743016
 psbAB+GFP+pPMQAK1
-De las cuales IntC no transformó. Se hizo colony PCR amplificando diversas partes de BBa_K743004  y psbAB+GFP+pPMQAK1, de los cuales se obtuvieron resultados positivos solo para BBa_K743004.
-Se picó e inoculo en matraz de 500ml BBa_K743014  para probar inducción por decanal. El inoculo creció en kanamicina pero no produjo lumiscencia en presencia de decanal.
+Only Int_C could not be transformed into cells. A colony PCR was run to amplify different sequences of BBa_K743004 and psbAB+GFP+pPMQAK1. Positive results for only for para BBa_K743004.
-Se recreció en placas de kanamicina BBa_K743004 y  psbAB+GFP+pPMQAK1 pero no crecieron. El constructo psbAB+GFP+pPMQAK1se plaqueo en kanamicina tres veces más, pero nunca creció, por lo que pensamos que realmente no transformo.
-Se transformo nuevamente Synechosystis pcc 6803 pero ahora es incubada en condiciones optimas (Luz continua y 30°C):
+BBa_K743014 was inoculated in a 500 mL flask to test induction by decanal. The inoculum grew in Kanamycin but did not produce bioluminescence when induced by decanal.
-BBa_K743004.
+BBa_K743004 and psbAB+GFP+pPMQAK1 was replated in Kanamycin plates but there was no growth. Construct psbAB+GFP+pPMQAK1 was replated in Kanamycin three times, but never grew. We believe it could not be transformed.
+We transformed Synechosystis pcc 6803 again with the following parts and now cells are being incubated at optimal conditions (continuous light and 30 °C).
+BBa_K743004
 BBa_K743009
 BBa_K743016
@@ Line 65: / Line 67: @@
 Control – (KAN)
 Control – (CLO)
-Nuevamente IntC no transformó. Pero el resto de las transformaciones parecen estar creciendo.
-Se plaquearon en placas de kanamicina BBa_K743014, BBa_K743016 desde las placas de transformación y se realizo un colony PCR para BBa_K743014 amplificando tanto LuxAB como LuxAB con su promotor.
-Se volvió a realizar una ronda de transformación de Synechosystis pcc 6803 con las mismas condiciones y constructos que la última.
+IntC could not be transformed again. The rest of the transformations seem ok.
-Las dos últimas transformaciones dieron controles negativos y colonias individuales para la mayoría de los constructos con excepción de IntC que no transformó. Se picaron colonias desde todas las trasformaciones y replaquearon en placas con kanamicina. En estas placas nuestras Synechosystys pcc 6803 crecieron en kanamicina, lo que nos confirma que existió transformación en ellas. Además, estas placas fueron sembradas en medio líquido con kanamicina donde también proliferaron.
-Luego de multiples replaqueos en kanamicina se realizo un Colony PCR masivo de las transformantes de las dos últimas rondas de transformación, donde tanto BBa_K743015 como BBa_K743016 confirmaron su transformación.
+BBa_K743014 and BBa_K743016 were plated in kanamycin plates from transformation plates and a colony PCR was run for BBa_K743014 amplifying LuxAB and LuxAB with promoter.
+A new round of Synechocystis was run under the same conditions and constructs that the last one.
+The last two transformations resulted in negative controls and individual colonies for most constrcuts except for IntC (did not transform). Colonies were picked from all transformations and were replated in kanamycin plates. Our cells grew in kanamycin, which indicates they are transformed. Also, the plates were inoculated I nliquid media with kanamycin and they also grew.
+After multiple replatings in Kanamycin, a massive colony PCR was run of the transformed cells from the last two rounds of transformation. BBa_K743015 and BBa_K743016 transformations were verified.
+Due to the important amount of failed attempts to transform IntC plasmid from Utah, this was digested resulting in bands of wrong size. We sent it to sequencing.
+Characterization of LuxBrick was done.
+A growth curve was characterized for Synechosystis PCC 6803 from an OD730:0.4800 inoculum.
+Inoculums:
+flasks with 1 mL in 150 mL of normal BG11.
+control with 1 mL in 150 mL of BG110.
+*Shaker  at 90 RPM.
+Experiment for characterization of Gibson for small parts was run.
+We tried to see LuxBrick induced under microscope. We concluded bioluminescence can’t be seen under microscope.
+We amplified parts and run a Gibson to assemble BBa_K743017 and BBa_K743018. Transformation and verification by digestion and PCR was done afterwards. Constructs were sent to sequence.
-Debido a la gran cantidad de intentos fallidos para transformar con el plásmido IntC de yhuta, este se digirió dando bandas de tamaños incorrectos, por lo que se mandó a secuenciar.
+Biolumiscence of different constructs with luciferase was measured when induced with different aldehides (decanal and dodecanal) using an Illumeter.
-Se creó un nuevo incubador de Synechosystis pcc 6803 a partir de un refrigerador en mal estado.
-Se realizo la Caracterización de LuxBrick (link?).
-Se realizó una curva de crecimiento (link?) para Synechosystis pcc 6803  a partir de un inoculo con OD730:0.4800. Se inocularon:
-matraces con 1ml de inoculo en 150ml de BG11normal.
-control con 1ml de inoculo en 150ml de BG110.
-*Shaker ~90 RPM.
-Se realizó el experimento para la caracterización de T5 (Gibson partes pequeñas).
-Se miraron LuxBrick inducidas bajo microscopio y se concluyo que estas no son visibles en microoscopía.
-Se amplificaron partes correspondientes y se procedió al Gibson y transformación de dos nuevos constructos: BBa_K743017 y BBa_K743018. Estos fueron validados mediante PCR y digestión, y se mandaron a secuenciar.
-Se realizo medición de bioluminiscencia con distintos aldheidos (decanal y dodecanal) para diferentes constructos con luciferasa mediante Iluminometro.
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