Team:UNIPV-Pavia/Calendar/June

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Revision as of 18:02, 17 June 2010

JUNE

DATE	ACTIVITY
June, 3th	9° Meeting - Discussion of final and technical details about The Project. Planning of Bio-Lab activity.
June, 7th	<partinfo>BBa_E2050</partinfo>, <partinfo>BBa_K165018</partinfo>, <partinfo>BBa_K165037</partinfo>, <partinfo>BBa_J61001</partinfo>, <partinfo>BBa_K081008</partinfo> and <partinfo>BBa_K125500</partinfo> were resuspended from Spring 2010 DNA distribution. <partinfo>BBa_P1004</partinfo> was resuspended from Spring 2009 DNA distribution. All BioBricks were transformed in E. coli DH5alpha. Liquid LB+Amp and LB+Amp agar plates were prepared according with "protocols".
June, 8th	Inoculum from plates in liquid Lb and glycerol stocks preparation. Falcon were re-filled for tomorrow mini-prep
June, 9th	MiniPrep of <partinfo>BBa_E2050</partinfo>, <partinfo>BBa_K165018</partinfo>, <partinfo>BBa_K165037</partinfo>, <partinfo>BBa_J61001</partinfo>, <partinfo>BBa_K081008</partinfo> and <partinfo>BBa_K125500</partinfo>. Digestion, gel run/cut and ligation of: I0: <partinfo>BBa_K125500</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X) I1: <partinfo>BBa_E2050</partinfo> (E-S)+<partinfo>BBa_K165018</partinfo> (E-X) I2: <partinfo>BBa_P1004</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X) I3: <partinfo>BBa_K081008</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
June, 10th	Transformation of ligations I0, I1, I2, I3 in home made E. coli DH5-alpha competent cells. Transformants were plated on LB+Amp agar plates
June, 11th	Inoculum from plates in liquid LB and glycerol stocks preparation for I0, I1, I2 and I3 in duplicate (2 colonies peaked from each plate).
June, 12th	iGEM EU workshop
June, 13th	iGEM EU workshop
June, 14th	6° Bio-Lab - Lorenzo and Nicolò.
June, 15th	7° Bio-Lab - Lorenzo and Manuel L., Sara and Alessandro.
June, 16th	8° Bio-Lab - Susanna Z., Sara and Nicolò.
June, 17th	9° Bio-Lab - Susanna Z., Nicolò and Riccardo.
June, 18th	10° Bio-Lab - Susanna Z., Susanna S. and Federica.

June, 7th

These BioBrick were resuspended in 15ul ddH20:

BBa_E2050 (mOrange, 744bp) 2010 Kit Plate 2, well 13N in pSB2K3
BBa_K165018 (ADH1 terminator, 253 bp) 2010 Kit Plate 3, well 2M, J63009 (Amp)
BBa_K165037 (tef2 promoter, 403bp) 2010 Kit Plate 3, well 22O, pSB1AK3
BBa_P1004 (Chloramphenicol resistence cassette, 769 bp) 2009 Kit plate 1, well 7B, pSB1A1
BBa_J61001 (R6K origin, 406bp) 2010 Kit plate 1, well 240, pSB1A2
BBa_K081008 (RBS-luxI, 664bp) 2010 Kit plate 2, well 10L, pSB1A2
BBa_K125500 (GFP fusion brick, 718bp) 2010 Kit plate 3, well 2P, pSB1A2

1,5 ul of each culture was transformed in 100 ul of home-made competent E. coli DH5alpha.

Tranformants were all plated on LB agar plates added with Ampicillin, except for BBa_E2050 (Kanamycin).

500 ml LB+Amp was prepared and 21 LB agar plates + Amp were prepared (500 ml).

June, 8th

All plates grown overnight at 37°C show colonies! In particular, <partinfo>BBa_E2050</partinfo> (gorwn on LB+Kan), <partinfo>BBa_K125500</partinfo> (gorwn on LB+Amp), <partinfo>BBa_K081008</partinfo> (gorwn on LB+Amp) and <partinfo>BBa_K165018</partinfo> (gorwn on LB+Amp) showed big colonies, well separated on the plate. <partinfo>BBa_P1004</partinfo> (gorwn on LB+Amp) and <partinfo>BBa_J61001</partinfo> (gorwn on LB+Amp) showed many colonies, with big colonies surrounded by small colonies. <partinfo>BBa_K165037</partinfo> (gorwn on LB+Amp) showed only 11 big colonies.

A colony was peaked for each plate and inoculated in 1ml LB+antibiotic.

<partinfo>BBa_E2050</partinfo>	1ml LB+Kan
<partinfo>BBa_K165037</partinfo>	1ml LB+Amp
<partinfo>BBa_P1004</partinfo>	1ml LB+Amp
<partinfo>BBa_K125500</partinfo>	1ml LB+Amp
<partinfo>BBa_K081008</partinfo>	1ml LB+Amp
<partinfo>BBa_J61001</partinfo>	1ml LB+Amp
<partinfo>BBa_K165018</partinfo>	1ml LB+Amp

Didascalia2

<partinfo>BBa_K165037</partinfo> in pSB1AK3 plasmid was inocultaed both in 1ml LB+Amp and in 1ml LB+Kan for a phenotipic assay.

Cultures were grown for 8 hours at 37°C 220 rpm.

After this time, cultures were all grown and in saturation phase.

Glycerol stocks were prepared for each culture (250ul saturated culture + 750 ul glycerol 80%) and stored at -80°C.

The left culture was re-filled to 5ml with LB+antibiotic and grown overnight at 37°C 200 rpm to be miniprepped tomorrow.

June, 9th

Ligation of:

I0: <partinfo>BBa_K125500</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
I1: <partinfo>BBa_E2050</partinfo> (E-S)+<partinfo>BBa_K165018</partinfo> (E-X)
I2: <partinfo>BBa_P1004</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
I3: <partinfo>BBa_K081008</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)

<partinfo>BBa_B0015</partinfo> was accidentally not inoculated yesterday... :( Luckily, we had a DNA stock for this part stored at -20°C, so we used this stock for ligations.

Cultures grown overnight at 37°C 220 rpm were all saturated. BioBricks were extracted with MiniPrep kit. Surprisingly, after the first centrifugation, we observed that <partinfo>BBa_E2050</partinfo> pellet was orange!! This is curious, because this BioBrick expresses mOrange, but it lacks of promoters. Probably, this spurious transcription is due to the promoter of Kanamycin resistance, that is oriented in the same direction.

BBa_E2050 orange pellet

After MiniPrep, purified DNA was quantified with NanoDrop.

<partinfo>BBa_E2050</partinfo>	139,2 ng/ul
<partinfo>BBa_K165037</partinfo>	181,5 ng/ul
<partinfo>BBa_P1004</partinfo>	132,8 ng/ul
<partinfo>BBa_K125500</partinfo>	124,3 ng/ul
<partinfo>BBa_K081008</partinfo>	138,3 ng/ul
<partinfo>BBa_J61001</partinfo>	122,2 ng/ul
<partinfo>BBa_K165018</partinfo>	166,3 ng/ul

Digestion of:

Culture	Kind	Final reaction volume (ul)	DNA (ul)	H20 (ul)	Enzyme 1	Enzyme 2	Buffer H
<partinfo>BBa_K125500</partinfo>	Insert	25	16	4,5	1 EcoRI	1 SpeI	2,5
<partinfo>BBa_B0015</partinfo>	Vector	25	6,3	14,2	1 EcoRI	1 Xba I	2,5
<partinfo>BBa_E2050</partinfo>	Insert	25	14	6,5	1 EcoRI	1 Spe I	2,5
<partinfo>BBa_K165018</partinfo>	Vector	25	6	14,5	1 EcoRI	1 Xba I	2,5
<partinfo>BBa_P1004</partinfo>	Insert	25	15	5,5	1 EcoRI	1 Spe I	2,5
<partinfo>BBa_K081008</partinfo>	Insert	25	14,5	6	1 EcoRI	1 Spe I	2,5

Digestions were incubated at 37°C for 3 hours, gel run and gel-extracted.

Gel run/cut of cultures

Ligation of I0, I1, I2 and I3 was performed at 16°C overnight.

June, 10th

I0, I1, I2 and I3 were tranformed in home made compotent E. coli DH5-alpha and plated on LB+Amp agar plates.

Plates with transformed E.coli were incubated overnight at 37°C 220 rpm.

File:UNIPV attivita lab4.jpg

The four plates were named:

iGEM 2010 10/06/2010 DH5-alpha I0 LB+Amp
iGEM 2010 10/06/2010 DH5-alpha I1 LB+Amp
iGEM 2010 10/06/2010 DH5-alpha I2 LB+Amp
iGEM 2010 10/06/2010 DH5-alpha I3 LB+Amp

and are stored at +4°C.

June, 11th

All four plates grown overnight showed colonies!! Two colonies for each plate were peaked and incubated in 1ml Lb+Amp. For I2, a phenotipic test was performed: the same two colonies were inoculated both in LB+Amp and LB+Cm. So, 10 cultures were incubated at 37°C 220 rpm for 8 hours:

I0-1 in 1ml LB+Amp;	I0-2 in 1ml Lb+Amp
I1-1 in 1ml LB+Amp;	I1-2 in 1ml Lb+Amp
I2-1 in 1ml LB+Amp;	I2-2 in 1ml Lb+Amp
I2-1 in 1ml LB+Cm;	I2-2 in 1ml Lb+Cm
I3-1 in 1ml LB+Amp;	I3-2 in 1ml Lb+Amp

After over-day growth, we observed that the phenotipic test for I2 was positive for both colonies: I2-1 and I2-1 were grown both in LB+Amp and LB+Cm.

8 glycerol stocks were prepared and stored at -80°C. Next week a screening will be performed to select positive colonies.

@@ Line 15: / Line 15: @@
 <tr><th align="center" width="12%"><b>DATE</b></th><th align="center"><b>ACTIVITY</b></th></tr>
 <tr><td align="center" valign="top">June, 3th</td><td align="left">9° Meeting - Discussion of final and technical details about [[Team:UNIPV-Pavia/Project|The Project]]. Planning of Bio-Lab activity.</td></tr>
-<tr><td align="center" valign="top">[[#June, 7th|June, 7th]]</td><td align="left"><partinfo>BBa_E2050</partinfo>, <partinfo>BBa_K165018</partinfo>, <partinfo>BBa_K165037</partinfo>, <partinfo>BBa_J61001</partinfo>, <partinfo>BBa_K081008</partinfo> and <partinfo>BBa_K125500</partinfo> were resuspended from Spring 2010 DNA distribution. <partinfo>BBa_P1004</partinfo> was resuspended from Spring 2009 DNA distribution. All BioBricks were transformed in E. coli DH5alpha.<br>Liquid LB+Amp and LB agar plates +Amp were prepared according with "protocols".</td></tr>
+<tr><td align="center" valign="top">[[#June, 7th|June, 7th]]</td><td align="left"><partinfo>BBa_E2050</partinfo>, <partinfo>BBa_K165018</partinfo>, <partinfo>BBa_K165037</partinfo>, <partinfo>BBa_J61001</partinfo>, <partinfo>BBa_K081008</partinfo> and <partinfo>BBa_K125500</partinfo> were resuspended from Spring 2010 DNA distribution. <partinfo>BBa_P1004</partinfo> was resuspended from Spring 2009 DNA distribution. All BioBricks were transformed in ''E. coli'' DH5alpha.<br>Liquid LB+Amp and LB+Amp agar plates were prepared according with "protocols".</td></tr>
 <tr><td align="center" valign="top">[[#June, 8th|June, 8th]]</td><td align="left">Inoculum from plates in liquid Lb and glycerol stocks preparation. Falcon were re-filled for tomorrow mini-prep<br></td></tr>
-<tr><td align="center" valign="top">[[#June, 9th|June, 9th]]</td><td align="left">MiniPrep of <partinfo>BBa_E2050</partinfo>, <partinfo>BBa_K165018</partinfo>, <partinfo>BBa_K165037</partinfo>, <partinfo>BBa_J61001</partinfo>, <partinfo>BBa_K081008</partinfo> and <partinfo>BBa_K125500</partinfo>. Digestion, gel run/cut and ligation of: *I0: <partinfo>BBa_K125500</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
+<tr><td align="center" valign="top">[[#June, 9th|June, 9th]]</td><td align="left">MiniPrep of <partinfo>BBa_E2050</partinfo>, <partinfo>BBa_K165018</partinfo>, <partinfo>BBa_K165037</partinfo>, <partinfo>BBa_J61001</partinfo>, <partinfo>BBa_K081008</partinfo> and <partinfo>BBa_K125500</partinfo>. Digestion, gel run/cut and ligation of:
+*I0: <partinfo>BBa_K125500</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
 *I1: <partinfo>BBa_E2050</partinfo> (E-S)+<partinfo>BBa_K165018</partinfo> (E-X)
 *I2: <partinfo>BBa_P1004</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
 *I3: <partinfo>BBa_K081008</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)<br></td></tr>
-<tr><td align="center" valign="top">[[#June, 10th|June, 10th]]</td><td align="left">Transformation of ligations I0, I1, I2, I3 in home made E. coli DH5-alpha competent cells. Transformants were plated on LB+Amp agar plates<br></td></tr>
+<tr><td align="center" valign="top">[[#June, 10th|June, 10th]]</td><td align="left">Transformation of ligations I0, I1, I2, I3 in home made ''E. coli'' DH5-alpha competent cells. Transformants were plated on LB+Amp agar plates<br></td></tr>
 <tr><td align="center" valign="top">[[#June, 11th|June, 11th]]</td><td align="left">Inoculum from plates in liquid LB and glycerol stocks preparation for I0, I1, I2 and I3 in duplicate (2 colonies peaked from each plate).<br></td></tr>
 <tr><td align="center" valign="top">June, 12th</td><td align="left">iGEM EU workshop<br></td></tr>
@@ Line 42: / Line 43: @@
 *BBa_K125500 (GFP fusion brick, 718bp) 2010 Kit plate 3, well 2P, pSB1A2
-,5 ul of each culture was transformed in 100 ul of home-made competent E. coli DH5alpha.
+,5 ul of each culture was transformed in 100 ul of home-made competent ''E. coli'' DH5alpha.
 Tranformants were all plated on LB agar plates added with Ampicillin, except for BBa_E2050 (Kanamycin).
@@ Line 151: / Line 152: @@
 </td>
 </tr>
-</table
+</table>
 Digestion of:
@@ Line 181: / Line 182: @@
 ==June, 10th==
-I0, I1, I2 and I3 were tranformed in home made compotent E. coli DH5-alpha and plated on LB+Amp agar plates.
+I0, I1, I2 and I3 were tranformed in home made compotent ''E. coli'' DH5-alpha and plated on LB+Amp agar plates.
-Transformed plates were incubated overnight at 37°C 220 rpm.
+Plates with transformed ''E.coli'' were incubated overnight at 37°C 220 rpm.
 <table align='center'><tr><td>
@@ Line 200: / Line 201: @@
 All four plates grown overnight showed colonies!!
-Two colonies for each plate were peaked and incubated in 1ml Lb+Amp. For I2, a phenotipic test was performed: the same two colonies were inoculated both in Lb+Amp and LB+Cm. So, 10 cultures were incubated at 37°C 220 rpm for 8 hours:
+Two colonies for each plate were peaked and incubated in 1ml Lb+Amp. For I2, a phenotipic test was performed: the same two colonies were inoculated both in LB+Amp and LB+Cm. So, 10 cultures were incubated at 37°C 220 rpm for 8 hours:
 {|
-| I0-1 in 1ml Lb+Amp;  ||    I0-2 in 1ml Lb+Amp
+| I0-1 in 1ml LB+Amp;  ||    I0-2 in 1ml Lb+Amp
 |-
-| I1-1 in 1ml Lb+Amp;  ||     I1-2 in 1ml Lb+Amp
+| I1-1 in 1ml LB+Amp;  ||     I1-2 in 1ml Lb+Amp
 |-
-| I2-1 in 1ml Lb+Amp;  ||      I2-2 in 1ml Lb+Amp
+| I2-1 in 1ml LB+Amp;  ||      I2-2 in 1ml Lb+Amp
 |-
-| I2-1 in 1ml Lb+Cm;  ||     I2-2 in 1ml Lb+Cm
+| I2-1 in 1ml LB+Cm;  ||     I2-2 in 1ml Lb+Cm
 |-
-| I3-1 in 1ml Lb+Amp;  ||     I3-2 in 1ml Lb+Amp
+| I3-1 in 1ml LB+Amp;  ||     I3-2 in 1ml Lb+Amp
 |}
-After over-day growth, we observed that the phenotipic test for I2 was positive for both colonies: I2-1 and I2-1 were grown both in Lb+Amp and LB+Cm.
+After over-day growth, we observed that the phenotipic test for I2 was positive for both colonies: I2-1 and I2-1 were grown both in LB+Amp and LB+Cm.
 glycerol stocks were prepared and stored at -80°C. Next week a screening will be performed to select positive colonies.

Team:UNIPV-Pavia/Calendar/June

From 2010.igem.org

Revision as of 18:02, 17 June 2010

June, 7th

June, 8th

June, 9th

June, 10th

June, 11th

June, 14th

June, 15th

June, 16th

June, 17th

June, 18th