From 2009.igem.org

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

• General Preparations:

1. chemiocompetent cells from E. Coli DH5α, Top10 and DB 3.1
2. antibiotic stocks of Ampicillin and Kanamicin
3. LB medium and LB plates
4. M9 medium

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

In order to realize our projects, we started looking for stardard plasmids.
We needed one high and one low to medium copy number vectors, with different antibiotic resistence (A & K). We searched the Registry and we decided two possible combinations: PSB1K3 with PSB4A3 or PSB1A2 with PSB3K3.

• Trasfomation of:

1. PSB1A2 -> with BBa_J23100 and BBa_J04031 from our parts library
2. PSB4A3 -> with BBa_J04500 and BBa_J04031 from our parts library

using Top10 chemically competent cells.

• Inoculation, miniprep preparation and control digestion, checked on agarose gel.
• Eluition and Trasfomation of:

1. PSB1K3 -> from 2009 kit, with BBa_J04450
2. PSB3K3 -> from 2009 kit, with BBa_J04450

using Top10 chemically competent cells.
Since neither PSB1K3 nor PSB3K3 yielded colonies we tried another transformation, choosing different kit wells.

• Eluition and Trasfomation 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 chemically competent cells.

• Inoculation, miniprep preparation and control digestion checked on agarose gel of PSB3K3 from 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 Registry.
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Week 3: from 08/03/09 to 08/07/09

• Annealing of Cis-repressing (CIS) and Trans-repressor (TRANS_4 and TRANS_7)
• E-P Digestion of:

1. CIS
2. TRANS_4
3. TRANS_7
4. PSB1A2

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 chemically competent cells
• Control 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 of our project circuit, 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 of 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 on 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 decide to study the different production of GFP 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 on DH5α chemically 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 chemically 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 of ligation on PSB4A3 revealed correct MW band (about 300bp), but the plasmid isolated from this colony revealed wrong MW after control 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 cheking on agarose gel.

Up

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

• We received primers for our sequences and, after some tests, we made PCR of our 3 parts. (We used as primers standard prefix and suffix sequences). We made PCR both from annealed parts that from single strands, and we choose to extract the latter, because they presented low 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

As the efficiency of restriction enzymes is considerably reduced when there aren’t enough bases near their restriction sites, we start 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 presents near restriction sites in all standard vectors.

In order to analyse the high/low copy number plasmid ratio we decide 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 assemblement of LacI repressor's natural operators on standard plasmids.

• Digestion of

1. K079017
2. K079018
3. K079019

• Ligation with RFP (BBa_E1010)
• Transformations on DH5α chemically competent cells
• Control digestion checked on agarose gel

• We received longer primers for our sequences. We made a new PCR of our 3 parts 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 3 standard BioBricks.

• Transformation on DH5α chemically competent cells
• Control 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 chemically competent cells
• Control 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
• PCR colony 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
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.

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