Team:Bologna/Lab-Notebook

From 2009.igem.org

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{{Template:BolognaTemplate}}
{{Template:BolognaTemplate}}
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= Week 1: from 07/20/09 to 07/24/09 =
= Week 1: from 07/20/09 to 07/24/09 =
-
In order to realize our projects, we started looking for stardard plasmids.  
+
* "Work Preparations":
-
We needed one high and one low to medium copy number vectors, with different antibiotic resistence (A & K).
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# chemiocompetent cells from E. Coli DH5α, Top10 and DB 3.1
-
We searched the Registry and we decided two possible combinations: PSB1K3 with PSB4A3 or PSB1A2 with PSB3K3.
+
# antibiotic stocks (Ampicillin and Kanamycin)
 +
# LB medium and plates
 +
# M9 medium
 +
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
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Trasfomation of:
+
= Week 2: from 07/27/09 to 07/31/09 =
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* PSB1K3 -> ''from 2009 kit, with BBa_J04450''
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* PSB3K3 -> ''from 2009 kit, with BBa_J04450''
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* PSB1A2 -> ''with BBa_J23100 and BBa_J04031''
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* PSB4A3 -> ''with BBa_J04500 and BBa_J04031''
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-
using Top10 chemically competent cells.
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-
 
+
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Since neither PSB1K3 nor PSB3K3 yielded colonies we tried another transformation, choosing different kit wells.
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-
 
+
-
Trasfomation of:
+
-
* PSB1K3 -> ''from 2009 kit, with BBa_P1010''
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* PSB3K3 -> ''from 2009 kit, with BBa_P1010''
+
-
* PSB3K3 -> ''from 2007 kit, with BBa_P1010''
+
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using DB3.1 chemically competent cells.
+
-
 
+
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Only PSB3K3 from 2007 kit worked correctly.
+
-
We decided to use PSB1A2 as high and PSB3K3 as low to medium copy number as they revealed right MW bands when checked with agarose gel.
+
-
In the meantime, we requested another PSB1K3 with BBa_P1010 from Registry.
+
 +
In order to build up our circuits, we started looking for stardard plasmids. <br>
 +
We needed one high and one medium copy number vector, with different antibiotic resistance (A & K).
 +
We searched the Registry and we chos two possible combinations: pSB1K3 with pSB4A3 or pSB1A2 with pSB3K3.
 +
*Transfomation of:
 +
# pSB1A2 -> ''with BBa_J23100 and BBa_J04031'' from our part library
 +
# pSB4A3 -> ''with BBa_J04500 and BBa_J04031'' from our part library
 +
using Top10 competent cells.
 +
*Inoculation, miniprep preparation and digestion, checked on agarose gel.
 +
*Eluition and Transfomation of:
 +
# pSB1K3 -> ''from 2009 kit, with BBa_J04450''
 +
# pSB3K3 -> ''from 2009 kit, with BBa_J04450''
 +
using Top10 competent cells.
 +
<br>
 +
Since neither pSB1K3 nor pSB3K3 yielded colonies, we tried another transformation, choosing different kit wells.
 +
*Eluition and Transfomation of:
 +
# pSB1K3 -> ''from 2009 kit, with BBa_P1010''
 +
# pSB3K3 -> ''from 2009 kit, with BBa_P1010''
 +
# pSB3K3 -> ''from 2007 kit, with BBa_P1010''
 +
using DB3.1 competent cells.
 +
*Inoculation, miniprep preparation and digestion checked on agarose gel of pSB3K3 (2007 kit).
 +
(the only one that yielded colonies)
 +
<br>
 +
We decided to use '''pSB1A2''' as high and '''pSB3K3''' as low to medium copy number as they revealed right MW bands when checked with agarose gel. In the meantime, we requested another pSB1K3 with BBa_P1010 from the Registry.
 +
<br>
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
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= Week 2: from 07/27/09 to 07/31/09 =
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= Week 3: from 08/03/09 to 08/07/09 =
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* Sequences suspension to have 100 μM conc. (EP digestion and trasformation in a HCN plasmid  with AMP)
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*Cis-repressing (CIS) and Trans-repressor (TRANS_4 and TRANS_7)annealing
 +
*E-P Digestion of:
 +
# CIS
 +
# TRANS_4
 +
# TRANS_7
 +
# PSB1A2
 +
<br>
{| border="1"
{| border="1"
! DNA sequences !! Plasmid
! DNA sequences !! Plasmid
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|'''TOT:''' 30  μM
|'''TOT:''' 30  μM
|'''TOT:''' 30  μM
|'''TOT:''' 30  μM
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|}
|}
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<br>
<br>
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'''Gel Run:'''
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[[Image:Notebook1.JPG|left|thumb|650px|CIS, TRANS_4, MARKER, TRANS_7]]
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'''1.'''CIS
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<br>
<br>
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'''2.'''TRANS 4
 
<br>
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'''3.'''MARKER
 
<br>
<br>
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'''4.'''TRANS 7
 
<br>
<br>
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'''5.'''PSB1A2
 
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<br><br>
 
-
[[Image:31_08_1a_CIS_T4_T7_PSB1A2.JPG|center|thumb|650px|CIS, TRANS 4, MARKER, TRANS 7, PSB1A2]]
 
-
 
-
 
-
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
 
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-
=Week 3: from 08/3/09 to 08/7/09=
 
-
 
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* Amplification of CIS, TRANS 4 and TRANS 7 using PSB1A2. Inoculation and digestion.
 
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* Unexpeted results from the gel run in all three sequences (3000, 2200, 1000 bp instead of 2100, 2000, 100 bp):
 
<br>
<br>
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[[Image:26_08_BandaMille.JPG|center|thumb|650px|Digestion of CIS, TRANS 4, TRANS 7 with unexpected 1000 bp insert]]
 
<br>
<br>
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* Done the ligation again whit the same bad results.
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<br>
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* Done some test fluorescence measurements using GFP in plasmids with different copy number.
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*Purification from agarose gel
 +
*Ligation of CIS, TRANS_4 and TRANS_7 on PSB1A2
 +
*Transformation on DH5α chemically competent cells (-> few colonies)
 +
*Inoculation and miniprep preparation
 +
*Control digestion checked on agarose gel revealed wrong MW bands
 +
<br>
 +
*Ligation of:
 +
# 2547_RBS_GFP_T on pSB1A2
 +
# 1429_RBS_GFP_T on pSB1A2
 +
# 2547_RBS_GFP_T on pSB3K3
 +
# 1429_RBS_GFP_T on pSB3K3
 +
*Transformation on DH5α competent cells
 +
*Digestion checked on agarose gel
 +
<br>
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
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HOLIDAY!!!
HOLIDAY!!!
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<br>
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[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
=Week 5: from 08/17/09 to 08/21/09=
=Week 5: from 08/17/09 to 08/21/09=
-
* Done again the amplifications of CIS, TRANS 4 and TRANS 7 using PSB1A2 to undestand which is the problem we have had during the third week. Achieved the same wrong results.  
+
In order to analyse the CIS/TRANS production ratio, we made some measures, using GFP as reporter.
-
* We decided to sequence the 1000 bp strand to understand what it really is. We sent our material to the Bmr-genomics in Padova for the dna sequencing.
+
*Transformation of
 +
# 1429_RBS_GFP_T on pSB3K3
 +
# 2547_RBS_GFP_T on pSB1A2
 +
# 1429_RBS_GFP_T on pSB3K3 + 2547_RBS_GFP_T on pSB1A2
 +
We made another cloning attempt for CIS, TRANS_4 and TRANS_7
 +
*X-P Digestion of:
 +
# CIS
 +
# TRANS_4
 +
# TRANS_7
 +
# PSB1A2
 +
# PSB4A3
 +
*Purification from agarose gel
 +
*Ligation of CIS, TRANS_4 and TRANS_7 both on pSB1A2 and pSB4A3
 +
*Transformation in DH5α chemically competent cells (-> few colonies)
 +
*Inoculation and miniprep preparation
 +
*Control digestion checked on agarose gel revealed wrong MW bands
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
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=Week 6: from 08/24/09 to 08/28/09=
=Week 6: from 08/24/09 to 08/28/09=
-
Bad news for our team:
+
In order to confirm the promoter ratio we decided to study the different GFP production without degradation tag (E0040) on pSB3K3 under J23100 (2547) and J23118 (1429)
 +
*Ligation of
 +
# 2547_I13504 on pSB3K3
 +
# 1429_I13504 on pSB3K3
 +
# 2547_I13504 on pSB1A2
 +
# 1429_I13504 on pSB1A2
 +
*Transformation in DH5α competent cells
 +
*Control digestion checked on agarose gel
 +
Experimental measures failed because GFP levels were too high to detect significant differences. We decided to use GFP with degradation tag (J04031 instead of E0040)
<br>
<br>
-
* Re-Annealing of our sequences and after have done the PRC-Colony we discovered that the colonies were green. Probably due to a GFP contamination. The DNA sequencing coming from Padova confirmed our theory: in the 1000  bp strand there was the presence of GFP protein and no sign of our CIS or Trans sequences.
+
*New annealing of CIS, TRANS_4 and TRANS_7
-
 
+
*Purification from agarose gel of the annealed parts
 +
*X-P Digestion of:
 +
# CIS
 +
# TRANS_4
 +
# TRANS_7
 +
# PSB1AK3
 +
*Purification from agarose gel
 +
*Ligation of CIS, TRANS_4 and TRANS_7 both on PSB1A2 and PSB4A3
 +
*Transformation on DH5α chemically competent cells
 +
*We checked colonies with colony PCR, obtaining wrong MW bands
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
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=Week 7: from 08/31/09 to 09/04/09=
=Week 7: from 08/31/09 to 09/04/09=
-
* Done some experiments (trasformation and digestion with the same plasmid, PSB1A2, using different concentration of annealed) to confirm the Gfp contamination. All the gel runs have a 1000 bp strand.
+
*Ligation of
-
* Investigation to discover where is the contamination.
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# 1429_RBS_LacI_T on PSB1A2
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* Starting use a new plasmid: PSB4A3. Trasfomation and digestion with the CIS sequence. From the gel run came out a 400 bp insert that could be correct (CIS+Primer=100bp+270bp=370bp):
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# 1429_RBS_LacI_T on PSB3K3
 +
*Transformation on DH5α chemically competent cells
 +
*Control digestion checked on agarose gel
<br>
<br>
-
[[Image:ColonyPCR_04_09cis_psb4a3_cis_psb3k3.JPG|center|thumb|650px|PCR Colony: CIS using PSB4A3 in the first well, CIS using PSB3K3 in the others ]]
+
*New X-P digestion of CIS annealed and purified through agarose gel, increasing DNA quantity
-
<br>
+
*Ligation both on PSB4A3 and PSB3K3
-
* The same attempt with TRANS 4 and TRANS 7 didn't give us any concrete results.
+
*Transformation on DH5α competent cells
 +
*We checked colonies with colony PCR
 +
[[Image:notebook2.JPG|left|thumb|650px|PCR Colony: CIS on PSB4A3 in the first well, CIS on PSB3K3 in the others ]]
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*Only one colony from the ligation on PSB4A3 revealed correct MW band (about 300bp), but the plasmid isolated from this colony revealed wrong MW after digestion checked on agarose gel
 +
<br>
 +
*Same results were obtained for TRANS_4 and TRANS_7 parts: colony PCRs seemed right, but we found wrong MW bands after checking on agarose gel.
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
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=Week 8: from 09/07/09 to 09/11/09=
=Week 8: from 09/07/09 to 09/11/09=
-
* Primers of our sequences arrived, so we amplified CIS-repressing and TRANS-repressor with a PCR. We designed primers so that they have the same sequence of the standard prefix and suffix.
+
* We received primers and we made PCR of our 3 parts. (We used as primers standard prefix and suffix sequences). We made PCR both from annealed parts and from single strands, and we chose to extract the latter, because of the smears when checked on agarose gel.
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* Gel run of results of PCR using a stain named GelStar that can detect smaller strains of DNA
+
[[Image:notebook3.JPG|left|thumb|580px|PCR of CIS-repressing and TRANS-repressor with primers]]
<br>
<br>
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[[Image:10_09_PCR.JPG|center|thumb|580px|PCR of CIS-repressing and TRANS-repressor with primers]]
 
<br>
<br>
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* We assumed that some of our ligation problems may be caused by the low enzymes efficiency because of the restriction sites were too near to the extremities of our sequeces. For that reason we decided to order longer primers in order to make our sequences longer and solve problems with enzymes.
+
<br>
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* Starting work with the operator Ox, developed by the [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Bologna 2008 Bologna Igem Team]
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* New digestion of CIS, TRANS_4 and TRANS_7 extracted from gel
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* Ligation on pSB1A2 yielded no colonies at all
 +
Since the restriction enzymes efficency is considerably reduced when there aren’t enough bases near their restriction sites, we started thinking that digestion could be our critical passage. We ordered longer primers, adding 7 bases to the end of each part. The added bases were the same for all standard vectors.  
 +
In order to analyse the high/low copy number plasmid ratio we decided to study the production of GFP without degradation tag on both pSB1A2 and pSB3K3, using 1429 promoter to avoid GFP saturation.
 +
Transformation of:
 +
# 1429_I13504 on pSB3K3
 +
# 1429_I13504 on pSB1A2
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
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=Week 9: from 09/14/09 to 09/18/09=
=Week 9: from 09/14/09 to 09/18/09=
-
* Primers arrived, so we can use them to make our sequences longer.
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We started to assemble LacI repressor's natural operators on standard plasmids.
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* The operator OX was mounted on a standard plasmid.
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*Digestion of
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* Tried a ligation with a plasmid containing the cell death gene, without positive results.
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#K079017
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#K079018
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#K079019
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*Ligation with RFP (BBa_E1010)
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*Transformations in DH5α chemically competent cells
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*Digestion checked on agarose gel
<br>
<br>
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[[Image:16_09_09.JPG|center|thumb|580px|PCR of CIS-repressing and TRANS-repressor with longer primers]]
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*We received longer primers for our sequences and made a new PCR in order to make them longer.
 +
*New cloning attempt failed again.
 +
<br>
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*Transformation of pSB1K3 received from Registry in order to try ligation with BBa_P1010 ("Death" gene)
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
-
 
=Week 10: from 09/21/09 to 09/25/09=
=Week 10: from 09/21/09 to 09/25/09=
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* Performed other tests with CIS-repressing and TRANS-repressor.  
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* New cloning attempts for CIS, TRANS_4 and TRANS_7. We reduced passage through gel using PCR extraction kit to purify PCR products and PSB1K3 with BBa_P1010
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* DNA-sequencing of the circuit with the operator OX, our first new BioBrick.
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*Digestion of
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#O2_RFP
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#O1_RFP
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#Oid_RFP
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and ligation on pSB1A2, in order to create standard BioBricks.
 +
*Transformation in DH5α competent cells
 +
*Digestion checked on agarose gel
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
=Week 11: from 09/28/09 to 10/02/09=
=Week 11: from 09/28/09 to 10/02/09=
 +
*Digestion of O2_RFP and ligation on:
 +
#PSB1A2_1429
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#PSB1A2_2547
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*Transformation on DH5α chemically competent cells
 +
*Control digestion checked on agarose gel
 +
 +
*Digestion of:
 +
#1429_O2_RFP on pSB1A2
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#2547_O2_RFP on pSB1A2
 +
and ligation with GFP (J04031)
 +
*Transformation on DH5α competent cells
 +
*Digestion checked on agarose gel
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* Ligation and mini-prep of the operator Ox, upstream of the protein GFP, under the control of the promoter J23118
 
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* Digestion and ligation of Cis-repressing and Trans-repressor using the restriction enzymes SpeI and XbaI.
 
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* Control digestion and Gel run; bur this test gave out only a strain, may be due to digestion problems. 
 
-
 
 
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
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=Week 12: from 10/05/09 to 10/09/09=
=Week 12: from 10/05/09 to 10/09/09=
 +
Thinking that our cloning problem was still due to wrong digestion, we made another attempt:
 +
* X-S digestion of TRANS_4 (we choose only one of our parts, since it was only a try)
 +
* Ligation on pSB1K3 with BBa_P1010
 +
*Transformation on DH5α chemically competent cells
 +
*Colony PCR made with two different primers revealed correct MW bands.
-
* Performed other tests mainly for perform fluorescence measures.
+
[[Image:picture3TS.jpg|left|thumb|580px|Colony PCR with standard Prefix and Suffix as primers: inserts were all at about 100bp as expected]]
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* Preparation of our BioBricks for the shipment.
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[[Image:picture4TS.jpg|left|thumb|580px|Colony PCR with standard VR and VF2 primers: inserts were all at about 300bp as expected (100bp is the length of our parts and 200bp are nucleotides due to primers’ binding on plasmid)]]
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[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
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=Week 13: from 10/12/09 to 10/16/09=
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<br>
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* Experimental measurement
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* Shipment of our parts to the Registry
 +
<br>
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[https://2009.igem.org/Team:Bologna/Lab-Notebook ''Up'']
 +
 +
More information about our CIS, TRANS_4 and TRANS_7 cloning attempt can be found in [https://2009.igem.org/Team:Bologna/T-REx_Story "Story of a T-REX"].
 +
<br>
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More information on experimental measures can be found in the [https://2009.igem.org/Team:Bologna/Characterization Characterization] section.

Latest revision as of 21:16, 21 October 2009

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HOME TEAM PROJECT SOFTWARE MODELING WET LAB PARTS HUMAN PRACTICE JUDGING CRITERIA


Contents

Week 1: from 07/20/09 to 07/24/09

  • "Work Preparations":
  1. chemiocompetent cells from E. Coli DH5α, Top10 and DB 3.1
  2. antibiotic stocks (Ampicillin and Kanamycin)
  3. LB medium and plates
  4. M9 medium

Up

Week 2: from 07/27/09 to 07/31/09

In order to build up our circuits, we started looking for stardard plasmids.
We needed one high and one medium copy number vector, with different antibiotic resistance (A & K). We searched the Registry and we chos two possible combinations: pSB1K3 with pSB4A3 or pSB1A2 with pSB3K3.

  • Transfomation of:
  1. pSB1A2 -> with BBa_J23100 and BBa_J04031 from our part library
  2. pSB4A3 -> with BBa_J04500 and BBa_J04031 from our part library

using Top10 competent cells.

  • Inoculation, miniprep preparation and digestion, checked on agarose gel.
  • Eluition and Transfomation of:
  1. pSB1K3 -> from 2009 kit, with BBa_J04450
  2. pSB3K3 -> from 2009 kit, with BBa_J04450

using Top10 competent cells.
Since neither pSB1K3 nor pSB3K3 yielded colonies, we tried another transformation, choosing different kit wells.

  • Eluition and Transfomation of:
  1. pSB1K3 -> from 2009 kit, with BBa_P1010
  2. pSB3K3 -> from 2009 kit, with BBa_P1010
  3. pSB3K3 -> from 2007 kit, with BBa_P1010

using DB3.1 competent cells.

  • Inoculation, miniprep preparation and digestion checked on agarose gel of pSB3K3 (2007 kit).

(the only one that yielded colonies)
We decided to use pSB1A2 as high and pSB3K3 as low to medium copy number as they revealed right MW bands when checked with agarose gel. In the meantime, we requested another pSB1K3 with BBa_P1010 from the Registry.
Up

Week 3: from 08/03/09 to 08/07/09

  • Cis-repressing (CIS) and Trans-repressor (TRANS_4 and TRANS_7)annealing
  • E-P Digestion of:
  1. CIS
  2. TRANS_4
  3. TRANS_7
  4. PSB1A2


DNA sequences Plasmid
H2O mQ == 4.5 μM H2O mQ == 19.5 μM
Sequence == 20 μM DNA (miniprep) == 5 μM
Buffer == 3 μM Buffer == 3 μM
BSA == 0.5 μM BSA == 0.5 μM
EcoRI enzyme == 1 μM EcoRI enzyme == 1 μM
Pst1 enzyme == 1 μM Pst1 enzyme == 1 μM
TOT: 30 μM TOT: 30 μM


CIS, TRANS_4, MARKER, TRANS_7

















  • Purification from agarose gel
  • Ligation of CIS, TRANS_4 and TRANS_7 on PSB1A2
  • Transformation on DH5α chemically competent cells (-> few colonies)
  • Inoculation and miniprep preparation
  • Control digestion checked on agarose gel revealed wrong MW bands


  • Ligation of:
  1. 2547_RBS_GFP_T on pSB1A2
  2. 1429_RBS_GFP_T on pSB1A2
  3. 2547_RBS_GFP_T on pSB3K3
  4. 1429_RBS_GFP_T on pSB3K3
  • Transformation on DH5α competent cells
  • Digestion checked on agarose gel


Up

Week 4: from 08/10/09 to 08/14/09

HOLIDAY!!!
Up

Week 5: from 08/17/09 to 08/21/09

In order to analyse the CIS/TRANS production ratio, we made some measures, using GFP as reporter.

  • Transformation of
  1. 1429_RBS_GFP_T on pSB3K3
  2. 2547_RBS_GFP_T on pSB1A2
  3. 1429_RBS_GFP_T on pSB3K3 + 2547_RBS_GFP_T on pSB1A2

We made another cloning attempt for CIS, TRANS_4 and TRANS_7

  • X-P Digestion of:
  1. CIS
  2. TRANS_4
  3. TRANS_7
  4. PSB1A2
  5. PSB4A3
  • Purification from agarose gel
  • Ligation of CIS, TRANS_4 and TRANS_7 both on pSB1A2 and pSB4A3
  • Transformation in DH5α chemically competent cells (-> few colonies)
  • Inoculation and miniprep preparation
  • Control digestion checked on agarose gel revealed wrong MW bands

Up

Week 6: from 08/24/09 to 08/28/09

In order to confirm the promoter ratio we decided to study the different GFP production without degradation tag (E0040) on pSB3K3 under J23100 (2547) and J23118 (1429)

  • Ligation of
  1. 2547_I13504 on pSB3K3
  2. 1429_I13504 on pSB3K3
  3. 2547_I13504 on pSB1A2
  4. 1429_I13504 on pSB1A2
  • Transformation in DH5α competent cells
  • Control digestion checked on agarose gel

Experimental measures failed because GFP levels were too high to detect significant differences. We decided to use GFP with degradation tag (J04031 instead of E0040)

  • New annealing of CIS, TRANS_4 and TRANS_7
  • Purification from agarose gel of the annealed parts
  • X-P Digestion of:
  1. CIS
  2. TRANS_4
  3. TRANS_7
  4. PSB1AK3
  • Purification from agarose gel
  • Ligation of CIS, TRANS_4 and TRANS_7 both on PSB1A2 and PSB4A3
  • Transformation on DH5α chemically competent cells
  • We checked colonies with colony PCR, obtaining wrong MW bands

Up

Week 7: from 08/31/09 to 09/04/09

  • Ligation of
  1. 1429_RBS_LacI_T on PSB1A2
  2. 1429_RBS_LacI_T on PSB3K3
  • Transformation on DH5α chemically competent cells
  • Control digestion checked on agarose gel


  • New X-P digestion of CIS annealed and purified through agarose gel, increasing DNA quantity
  • Ligation both on PSB4A3 and PSB3K3
  • Transformation on DH5α competent cells
  • We checked colonies with colony PCR
PCR Colony: CIS on PSB4A3 in the first well, CIS on PSB3K3 in the others














  • Only one colony from the ligation on PSB4A3 revealed correct MW band (about 300bp), but the plasmid isolated from this colony revealed wrong MW after digestion checked on agarose gel


  • Same results were obtained for TRANS_4 and TRANS_7 parts: colony PCRs seemed right, but we found wrong MW bands after checking on agarose gel.

Up

Week 8: from 09/07/09 to 09/11/09

  • We received primers and we made PCR of our 3 parts. (We used as primers standard prefix and suffix sequences). We made PCR both from annealed parts and from single strands, and we chose to extract the latter, because of the smears when checked on agarose gel.
PCR of CIS-repressing and TRANS-repressor with primers













  • New digestion of CIS, TRANS_4 and TRANS_7 extracted from gel
  • Ligation on pSB1A2 yielded no colonies at all

Since the restriction enzymes efficency is considerably reduced when there aren’t enough bases near their restriction sites, we started thinking that digestion could be our critical passage. We ordered longer primers, adding 7 bases to the end of each part. The added bases were the same for all standard vectors.

In order to analyse the high/low copy number plasmid ratio we decided to study the production of GFP without degradation tag on both pSB1A2 and pSB3K3, using 1429 promoter to avoid GFP saturation. Transformation of:

  1. 1429_I13504 on pSB3K3
  2. 1429_I13504 on pSB1A2

Up

Week 9: from 09/14/09 to 09/18/09

We started to assemble LacI repressor's natural operators on standard plasmids.

  • Digestion of
  1. K079017
  2. K079018
  3. K079019
  • Ligation with RFP (BBa_E1010)
  • Transformations in DH5α chemically competent cells
  • Digestion checked on agarose gel


  • We received longer primers for our sequences and made a new PCR in order to make them longer.
  • New cloning attempt failed again.


  • Transformation of pSB1K3 received from Registry in order to try ligation with BBa_P1010 ("Death" gene)

Up

Week 10: from 09/21/09 to 09/25/09

  • New cloning attempts for CIS, TRANS_4 and TRANS_7. We reduced passage through gel using PCR extraction kit to purify PCR products and PSB1K3 with BBa_P1010
  • Digestion of
  1. O2_RFP
  2. O1_RFP
  3. Oid_RFP

and ligation on pSB1A2, in order to create standard BioBricks.

  • Transformation in DH5α competent cells
  • Digestion checked on agarose gel

Up

Week 11: from 09/28/09 to 10/02/09

  • Digestion of O2_RFP and ligation on:
  1. PSB1A2_1429
  2. PSB1A2_2547
  • Transformation on DH5α chemically competent cells
  • Control digestion checked on agarose gel
  • Digestion of:
  1. 1429_O2_RFP on pSB1A2
  2. 2547_O2_RFP on pSB1A2

and ligation with GFP (J04031)

  • Transformation on DH5α competent cells
  • Digestion checked on agarose gel

Up

Week 12: from 10/05/09 to 10/09/09

Thinking that our cloning problem was still due to wrong digestion, we made another attempt:

  • X-S digestion of TRANS_4 (we choose only one of our parts, since it was only a try)
  • Ligation on pSB1K3 with BBa_P1010
  • Transformation on DH5α chemically competent cells
  • Colony PCR made with two different primers revealed correct MW bands.
Colony PCR with standard Prefix and Suffix as primers: inserts were all at about 100bp as expected













Colony PCR with standard VR and VF2 primers: inserts were all at about 300bp as expected (100bp is the length of our parts and 200bp are nucleotides due to primers’ binding on plasmid)































Up

Week 13: from 10/12/09 to 10/16/09


  • Experimental measurement
  • Shipment of our parts to the Registry


Up

More information about our CIS, TRANS_4 and TRANS_7 cloning attempt can be found in "Story of a T-REX".

More information on experimental measures can be found in the Characterization section.

Retrieved from "http://2009.igem.org/Team:Bologna/Lab-Notebook"