Team:TU Darmstadt/Labjournal/Transport

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Revision as of 16:48, 18 September 2012

Transport

1 Week 1 (21.-25.05.12)
2 week 2 (28.05.-01.06.12)
3 week 3 (04.-08.06.12)
4 week 4 (11.-15-06.12)
5 week 5 (18.-22.06.12)
6 week 6 (25.-29.06.12)
7 week 7 (02.-06.07.12)
8 week 8 (09.-13.07.12)
9 week 9 (16.-20.07.12)
10 week 10 (23.-27.07.12)
11 week 11 (30.07.-03.08.12)
12 week 12 (06.-10.08.12)
13 week 13 (13.-17.08.12)
14 week 14 (20.-24.08.12)
15 week 15 (27.-31.08.12)

Week 1 (21.-25.05.12)

First colony PCR of Comamonas testosteroni for isolation of the following genes: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tctB 197aa] (2), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 tctA_503aa] (3), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tctB 162aa] (4), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5). (Primer: tctA_503 Biobrick, tctA_505 Biobrick, tctB_162 Biobrick, tctB_197 Biobrick, tphC Biobrick). Analysis of [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] products by Agarose gel electrophoresis

PCR product purification using the Promega-Kit

Nanodrop measurements of the purified PCR products:

(1) : 19.5 ng/µl 260/280=1.58

(2) : 61.9 ng/µl 260/280=1.75

(3) : 75.1 ng/µl 260/280=1.75

(4) : 93.3 ng/µl 260/280=1.85

(5) : 91.3 ng/µl 260/280=1.58

Restriction digest of PCR products by SpeI and EcoRI, heat inactivation after digestion

Analysis of restriction digest by Agarose gel electrophoresis

Purification of the bands gained form gel electrophoresis (Promega-Kit)

Nanodrop measurements of the purified products:

(1) : 8.3 ng/µl 260/280=1.94

(2) : 8.9 ng/µl 260/280=1.80

(3) : 13.0 ng/µl 260/280=1.57

(4) : 1.5 ng/µl 260/280=3.08

(5) : 3.7 ng/µl 260/280=2.26

week 2 (28.05.-01.06.12)

Purified products from 1. week were ligated into pSB1A2 and subsequently transformed into DH5a (in spite of low concentration)

Overnight incubation on LB agar plates; no growth detectable

Second approach to isolate the five genes from Comamonas t. using colony-PCR (Primer: tctA_503 Biobrick, tctA_505 Biobrick, tctB_162 Biobrick, tctB_197 Biobrick, tphC Biobrick)

Analysis of PCR products by Agarose gel electrophoresis; without results; modification of the PCR protocols: adding DMSO

Third approach to isolate the five genes from Comamonas t. by colony-PCR (Primer: tctA_503 Biobrick, tctA_505 Biobrick, tctB_162 Biobrick, tctB_197 Biobrick, tphC Biobrick)

PCR product purification using the Promega-Kit: ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tctB 197aa] (2), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 tctA_503aa] (3), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tctB 162aa] (4), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5))

(1): no PCR-product

Nanodrop measurements of the purified PCR products:

(2) : 56.1 ng/µl 260/280=1.87

(3) : 66.8 ng/µl 260/280=1.92

(4) : 72.8 ng/µl 260/280=1.87

(5) : 68.8 ng/µl 260/280=1.82

week 3 (04.-08.06.12)

Restriction digest of the purified PCR products and of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (leftover of the 1. week) by SpeI and EcoRI

Restriction digest of pSB1A2 using SpeI and EcoRI

Dephosphorylation of the restriction reactions by using antarctic Phosphatase

Ligation of the five genes into pSB1A2 and subsequently transformation into DH5a (incubation at 37°C)

colony-PCR of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1), 2 approaches (Primer:tctA_505 Biobrick)

Analysis of PCR products by Agarose gel electrophoresis. Only one of them showed the expected band. The band was excluded and purified via the Promega-Kit.

Nanodrop measurement of the purified product:

(1.1) =8.9 ng/µl 260/280=2,22

Because of low concentration and contamination the PCR was repeated and the product was purified again by the Promega-Kit.

Performing a colony-PCR-screen of the transformed cells using the Taq-Polymerase. Verification of 4 colonies per plate. (Primer: tctA_503 Biobrick, tctA_505 Biobrick, tctB_162 Biobrick, tctB_197 Biobrick, tphC Biobrick)

PCR did not work

Testing the Taq-Polymerase for function. The Phu-Polymerase was used for comparison. Only the approach using the Phu-Polymerase showed the expected bands in Agarose gel electrophoresis.

Screening was repeated with Phu-Polymerase; no bands visible

week 4 (11.-15-06.12)

Inoculation of LB-media with DH5a_pSB1A2 and DH5a_pSB1C3; overnight incubation at 37°C

Miniprep of DH5a_pSB1A2 and DH5a_pSB1C3 using the Promega-Kit

Nanodrop measurements of the preparation:

pSB1A2 : 136.1 ng/µl 260/280= 1.93

pSB1C3 : 265.1 ng/µl 260/280= 1.89

Restriction digest of pSB1A2 and pSB1C3 with EcoRI and SpeI. Afterwards dephosphorylation by using Antarctic phosphatase.

Analysis of the restriction products through Agarose gel electrophoresis; for comparison the uncropped vectors were also analyzed.

pSB1C3 shows several bands (Insert, vector without insert, linearized vector with insert); concentration of pSB1A2 is very low, only one band visible

Approach to isolate the following genes from Comamonas t. by colony-PCR: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tctB 197aa] (2), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 tctA_503aa] (3), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tctB 162aa] (4) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5) by [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] (Primer: tctA_503 Biobrick, tctA_505 Biobrick, tctB_162 Biobrick, tctB_197 Biobrick, tphC Biobrick)

Analysis of PCR products by Agarose gel electrophoresis

Isolation of (1) and (5) did not work

PCR product purification by using the Promega-Kit

Nanodrop measurements of the purified PCR products:

(2) : 18.3 ng/µl 260/280=2.03

(3) : 21.2 ng/µl 260/280=1.95

(4) : 23.9 ng/µl 260/280=2.05

Restriction digest of purified PCR products (2), (3) and (4) using SpeI and EcoRI

Ligation of the digested genes ((2), (3), (4)) into pSB1A2 and pSB1C3

Transformation of DH5a with pSB1A2_197, pSB1A2_503, pSB1A2_162, pSB1C3_197, pSB1C3_503, pSB1C3_162 (6 different transformation approaches). Overnight incubation at 37°C

Screening of the transformants by colony-PCR (Primer: tctA_503 Biobrick, tctA_505 Biobrick, tctB_162 Biobrick, tctB_197 Biobrick, tphC Biobrick). Verification of 6 colonies for every transformation. Afterwards the PCR products were analyzed by Agarose gel electrophoresis

positive colonies are marked

Colony-PCR of Comamonas t. to isolate [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1) und [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5) (Primer: tctA_505 Biobrick, tphC Biobrick)

Analysis of PCR products (Colony-PCR) and the ligation approach by Agarose gel electrophoresis

PCR of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] shows only the band of the undesired smaller product

Restriction digest of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5) and subsequent Ligation into pSB1A2 and pSB1C3

Transformation of DH5a with pSB1A2_322 und pSB1C3_322. [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] (Primer: tphC Biobrick) used to check for successful transformation

Isolation of Comamonas t. genome, because it seems impossible to amplificate [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1) from a fluid Comamonas t. culture.

week 5 (18.-22.06.12)

Introduction of a nomenclature: A/C gene/colony: pSB1A2 = A; pSB1C3 = C; gene: tctA 505aa = 1, tctB 197aa = 2, tctA 503aa = 3, tctB 162aa = 4, tphC 322aa = 5; colony = 1...X

Producing fluid cultures of the positive transformants

Miniprep of the fluid culures by using the Frementas-Kit. Afterwards Nanodrop measurements of the preparations

A2.3 : 125.9 ng/µl 260/280= 1.62

A3.1 : 103.3 ng/µl 260/280= 1.72

A4.6 : 147.2 ng/µl 260/280= 1.70

A4.5 : 108.7 ng/µl 260/280= 1.86

A5.12 : 88.0 ng/µl 260/280= 1.87

A5.8 : 85.01 ng/µl 260/280= 1.78

A3.2 : 87.3 ng/µl 260/280= 1.81

C4.6 : 70.5 ng/µl 260/280= 1.81

C4.1 : 41.2 ng/µl 260/280= 1.85

C4.4 : 104.2 ng/µl 260/280= 1.85

C2.1 : 132.2 ng/µl 260/280= 1.85

C2.6 : 188.1 ng/µl 260/280= 1.75

[http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] using a rest of purified [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1) (Primer: tctA_505 Biobrick), subsequent Agarose gel electrophoresis

Another colony-PCR using the transformants (2)-(5) as template (Primers: tctA_503 Biobrick, tctB_162 Biobrick, tctB_197 Biobrick, tphC Biobrick); analyzing the PCR product by Agarose gel electrophoresis

3 positive transformants were picked for inoculation of fluid media (A34, A43; C513)

Miniprep (Fermentas Kit) of the fluid culures and Nanodrop measurements:

A2.4 : 202,1 ng/µl 260/280= 1,63

A2.5 : 126,5 ng/µl 260/280= 1,90

A2.6 : 107,5 ng/µl 260/280= 1,81

A4.2 : 133,8 ng/µl 260/280= 1,59

C2.3 : 187,8 ng/µl 260/280= 1,87

C2.4 : 270,7 ng/µl 260/280= 1,80

C4.1 : 103,8 ng/µl 260/280= 1,80

The Miniprep products were Restriction digested by restriction enzymes, each was cut once with EcoRI and twice with EcoRI/SpeI. Analisys of the digested products by [Agarose gel electrophoresis

week 6 (25.-29.06.12)

Further colony-PCR of Comamonas t. (3 approaches) and [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] with genomic Comamonas t. DNA as template to obtain [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1), 2 approaches each (Primer: tctA_505 Biobrick). Subsequent analisys of the PCR products by Agarose gel electrophoresis. All PCR of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] show only the band of the undesired smaller product.

Ligation off the digested genes into pSB1A2 and transformation into DH5a. Overnight incubation at 37°C

Colony-PCR of Comamonas t. and [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] with genomic Comamonas t. DNA as template to obtain [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1). [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tctB 197aa] (2), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 tctA_503aa] (3), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tctB 162aa] (4), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5) used as control (Primers: tctA_503 Biobrick, tctA_505 Biobrick, tctB_162 Biobrick, tctB_197 Biobrick, tphC Biobrick)

Miniprep and Nanodrop measurement of:

A3.1 : 111.6 ng/µl 260/280= 1.72

A3.4 : 79.5 ng/µl 260/280= 1.66

C4.6 : 69.6 ng/µl 260/280= 1.76

A4.2 : 96.9 ng/µl 260/280= 1.62

A2.4 : 109.7 ng/µl 260/280= 1.72

A2.5 : 160.8 ng/µl 260/280= 1.79

A5.13 : 188.7 ng/µl 260/280= 1.85

A5.12 : 114.5 ng/µl 260/280= 1.81

Checking the Miniprep with [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] (Primers: tctA_503 Biobrick, tctB_162 Biobrick, tctB_197 Biobrick, tphC Biobrick) and Agarose gel electrophoresis

Colony-PCR-Screen of the transformants (Primer: tctA_505 Biobrick) and analisys of the PCR products by Agarose gel electrophoresis

No bands visible

Ligation of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] into pSB1C3 and subsequently transformation into DH5a. No colonies

The following vectors were sequenced: pSB1A2_197.4, pSB1A2_197.5, psSB1A2_503.1, pSB1A2_503.4, pSB1A2_162.2, pSB1C3_162.6, pSB1A2_322.12, pSB1A2_322.13

Restriction digest of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] using EcoRI/SpeI. Ligation into pSB1C3 and subsequently transformation into DH5a.

Preperations for pure culture: colony-PCR of A2.4, A2.5, A3.1, A3.4, A4.2, C4.6, A5.12, A5.13 (2 colonies each, Primers: tctA_503 Biobrick, tctB_162 Biobrick, tctB_197 Biobrick, tphC Biobrick) and afterwards checking the PCR products by Agarose gel electrophoresis. Positive colonies were picked, streaked onto fresh media and incubated again.

As A5.12 and A5.13 are missing the right insert the colony-PCR was repeated (5 and 6 colonies were used, Primer: tphC Biobrick). To provide additional monitoring the colony-PCR was repeated for A2.5_2, A3.1_2 und A4.2_2. 2 positive colonies were again streaked onto fresh media.

The Arabinose-Promotor ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara]) was isolated from pbad_turbo_gfp and colonies containing this plasmid (3 approaches each). Approach T2 is further in use.

week 7 (02.-06.07.12)

Restriction digest of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] and pXylE using EcoRI and SpeI. Ligation of the restricted products and transformation into DH5a. No colonies

Restriction digest of pSB1A2 using EcoRI and SpeI

DH5a-cells containing the vectors A4.2_2, A3.1_2, A2.5_2, A5.13_5 were diluted in glycerol (20%) and freezed (-80°C).

Mutagenic PCR of A4.2, A5.13, A3.1 to eliminate the PstI restriction site (Primers: tctA_503_PstI, tctB_162_PstI, tphC_PstI). Transformation into DH5a
Checking the DH5a-cells for correct transformation with the vectors after the mutagenic PCR by colony-PCR (checking 7 colonies each, Primers: tctA_503 Biobrick, tctB_162 Biobrick, tphC Biobrick). The PCR products were digested with PstI. The gel was alternately loaded with digested and uncut PCR products.

The transformation of DH5a with pSB1C3_tctA_505aa was verified by colony-PCR (Primers: tctA_505 Biobrick). The positive colonies were used for Miniprep

Miniprep (Fermentas Kit) and Nanodrop measurement:

A5.13m_1 : 105.2 ng/µl 260/280= 1.84

A5.13m_2 : 223.0 ng/µl 260/280= 1.87

A5.13m_3 : 110.6 ng/µl 260/280= 1.87

A5.13m_4 : 99.6 ng/µl 260/280= 1.90

A5.13m_6 : 326.6 ng/µl 260/280= 1.88

A5.13m_7 : 145.0 ng/µl 260/280= 1.84

A3.1m_1 : 241.7 ng/µl 260/280= 1.87

A3.1m_2 : 188.4 ng/µl 260/280= 1.89

A3.1m_3 : 112.4 ng/µl 260/280= 1.89

A3.1m_4 : 173.1 ng/µl 260/280= 1.85

A3.1m_5 : 111.6 ng/µl 260/280= 1.85

A3.1m_6 : 138.1 ng/µl 260/280= 1.87

A3.1m_7 : 178.9 ng/µl 260/280= 1.86

A4.2m_1 : 57.0 ng/µl 260/280= 1.84

A4.2m_2 : 46.5 ng/µl 260/280= 1.77

A4.2m_3 : 55.7 ng/µl 260/280= 1.88

A4.2m_4 : 111.3 ng/µl 260/280= 1.82

A4.2m_5 : 236.9 ng/µl 260/280= 1.89

A4.2m_6 : 69.3 ng/µl 260/280= 1.89

A4.2m_7 : 72.3 ng/µl 260/280= 1.85

C1.2 : 132.9 ng/µl 260/280= 1.72

new transformation of DH5a with pXylE_Ara

week 8 (09.-13.07.12)

Checking the transformation of DH5a with pSB1C3_tctA_505aa again (colony-PCR) [*]. The colonies Nr. 2, 8 and 10 were picked and streaked onto fresh media.

colony-PCR of DH5a_pXylE_Ara [*]

No positive colonies

Sequencing the following vectors: pSB1A2_4.2m_5, pSB1A2_4.2m_4, pSB1A2_5.13m_3, pSB1A2_5.13m_2, pSB1A2_3.1m_1, pSB1A2_3.1m_5, pSB1A2_3.1m_7

Checking the transformation of DH5a with A1 (5 colonies) and C1 (7 colonies) by using Colony-PCR [?]

Checking the transformation of DH5a with pSB1C3_tctA_505aa again by colony-PCR. Only the colonies Nr. 2, 8 and 10 (see above) were checked (2 colonies each).

Another colony-PCR of DH5a_pXy19_Ara

Miniprep (Fermentas Kit) and Nanodrop measurement of:

A5.13m_2 : 191.2 ng/µl 260/280= 1.84

A5.13m_3 : 168.1 ng/µl 260/280= 1.78

A2.5 : 193.0 ng/µl 260/280= 1.83

A3.1m_5 : 146.1 ng/µl 260/280= 1.82

A3.1m_1 : 250.1 ng/µl 260/280= 1.84

A4.2m_4 : 124.8 ng/µl 260/280= 1.85

Restriction digest of A5.13m_2, A5.13m_3, A3.1m_1, A3.1m_5, A2.5, A4.2m_4 with EcoRI and SpeI. The restriction was done to enable the ligation of the mutated genes into pSB1C3. Afterwards DH5a was transformed with the ligation product.

Miniprep (Fermentas Kit) and Nanodrop measurement of:

C505_2_2 : 253.6 ng/µl 260/280= 1.60

C505_2_3 : 274.7 ng/µl 260/280= 1.88

C505_8_2 : 462.1 ng/µl 260/280= 1.38

C505_8_3 : 149.1 ng/µl 260/280= 1.84

C505_10_3 : 160.4 ng/µl 260/280= 1.98

Restriction digest of the Miniprep products with EcoRI and EcoRI/SpeI. Afterwards the restriction products were analyzed by Agarose gel electrophoresis. Gel loading: uncut, cut once, cut twice

C505_2_3 und C505_8_2 were selected for mutagenic PCR.

Restriction digest of pSB1C3 with EcoRI and SpeI

Restriction digest of pXylE with EcoRI and XbaI and of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] with EcoRI and SpeI. Dephosphorylation of pXylE. Ligation of pXylE and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara]

week 9 (16.-20.07.12)

mutagenic PCR of C505_2_3 and C505_8_2. (Did not work)

[http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tct_B162m] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tct_B197m] with primer to add a RBS in front and behind the gene

Restriction digest of the PCR products with EcoRI and SpeI for ligation into pSB1C3_5.13m (tphC)

Adding a RBS to one site of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] using PCR with the reverse primer

Miniprep (Fermentas Kit) and Nanodrop measurement of:

C3.1m_1 : 278.9 ng/µl 260/280= 1.88

C4.2m_4 : 214.2 ng/µl 260/280= 1.86

5.13m_3 : 256.3 ng/µl 260/280= 1.88

C2.5_2 : 44.8 ng/µl 260/280= 2.00

C5.13m_2 : 60.8 ng/µl 260/280= 1.89

week 10 (23.-27.07.12)

Restriction digest of pSB1C3_5.13m_3_2 and pSB1C3_5.13m_3_3 with EcoRI and XbaI. Afterwards the products are ligated with RBS_tctB162m_RBS and RBS_tctB197_RBS each.

Restriction digest of A3.1m_1 for ligation with Ara

Digestion of pXylE for ligation with Ara_RBS

[http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] of Ara_RBS and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] for ligation into pXylE and pSB1C3. Afterwards restriction digest with restriction enzymes: Ara_RBS is cut with with EcoRI and SpeI and Ara is cut with EcoRI and XbaI

Ligation of the restricted PCR-products to: pSB1A2_tctB197_tphCm, pSB1C3_tctB162m_tphCm, pSB1C3_tct197_tphCm, pSB1A2_tctB162m_tphCm, pSB1A2_Ara_tctA503m, pSB1C3_Ara_tctA503m, pSB1A2_Ara_XylE and transformation into DH5a

mutagenic PCR of 505_8_2

week 11 (30.07.-03.08.12)

Checking transformation of DH5a by colony-PCR: pSB1A2_tctB197_tphCm, pSB1C3_tctB162m_tphCm, pSB1C3_tct197_tphCm, pSB1A2_tctB162m_tphCm, pSB1A2_Ara_tctA503m, pSB1C3_Ara_tctA503m, pSB1A2_Ara_XylE. Positive colonies are picked and used for inoculation of fluid media.

Miniprep (Fermentas Kit) of fluid culture and Nanodrop measurement of:

pSB1C3_503 : 110.0 ng/µl 260/280= 1.82

pSB1A2_tctB197_tphCm : 32.0 ng/µl 260/280= 2.10

pSB1A2_Ara_tctA503m : 202.0 ng/µl 260/280= 1.78

pSB1A2_tctB162m_tphCm : 36.5 ng/µl 260/280= 1.91

pSB1C3_Ara_tctA503m : 133.2 ng/µl 260/280= 1.83

pSB1C3_tctB162m_tphCm : 35.2 ng/µl 260/280= 1.93

pSB1C3_tct197_tphC : 30.6 ng/µl 260/280= 1.88

Restriction digest of pSB1A2_Ara_tctA503m, pSB1C3_Ara_tctA503m, pSB1A2_tctB162m_tphCm and pSB1C3_tctB162m_tphCm and building the final constructs by ligation: pSB1A2_tctA503m_tctB162m_tphCm und pSB1C3_tctA503m_tctB162m_tphCm. Transformation of the final constructs into DH5a.

Transformation of pSB1C3_503 into DH5a

Checking the mutagenic PCR product of 505_8_2 and the transformation of DH5a with pSB1C3_503 by [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR]

Restriction digest of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] und C4.2_4_1 using EcoRI and PstI, building pSB1C3_Ara by ligation and transformation of the vector into DH5a

Miniprep (Fermentas Kit) and Nanodrop measurement of:

pSB1C3_Ara : 86.7 ng/µl 260/280= 1.83

pSB1C3_505m : 113.8 ng/µl 260/280= 1.85

Restriction digest of pSB1C3_505m with PstI. Checking if the restriction site has been successfully removed

restriction site hasn't been removed

Screnning for transformation of DH5a with pSB1A2_tctA503m_tctB162m_tphCm and pSB1C3_tctA503m_tctB162m_tphCm using colony-PCR [*]

Checking the constructs pSB1A2/pSB1C3_Ara_503m and pSB1A2/pSB1C3_tctB162m_tphCm by [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] [*]

Ligation of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] into pSB1C3

Checking if pSB1A2_tctB162m_tphCm and pSB1C3_tctB162m_tphCm have the right Prefix/Suffix by digesting both with EcoRI and XbaI and with EcoRI and PstI [*]

Ligation of pSB1A2_tctB162m_tphCm and pSB1C3_tctB162m_tphCm with Ara_503

Miniprep (Fermentas Kit) and Nanodrop measurement of:

pSB1A2_tctB162m_tphCm_1 : 45.3 ng/µl 260/280= 1.90

pSB1A2_tctB162m_tphCm_2 : 74.5 ng/µl 260/280= 1.82

pSB1C3_tctB162m_tphCm_1 : 95.8 ng/µl 260/280= 1.86

pSB1C3_tctB162m_tphCm_2 : 85.7 ng/µl 260/280= 1.81

pSB1C3_tctB162m_tphCm_3 : 100.1 ng/µl 260/280= 1.84

pSB1C3_tctB162m_tphCm_4 : 79.9 ng/µl 260/280= 1.88

pSB1C3_tctB162m_tphCm_5 : 89.8 ng/µl 260/280= 1.85

Sequencing pSB1A2_tctB162m_tphCm_2 and pSB1C3_tctB162m_tphCm_5

week 12 (06.-10.08.12)

Miniprep (Fermentas Kit) and Nanodrop measurement of:

pSB1A2_tctB162m_tphCm_2 : 55.7 ng/µl 260/280= 1.81

pSB1C3_tctB162m_tphCm_5 : 101.2 ng/µl 260/280= 1.92

Mutagenic PCR of pSB1C3_505_8_2 and subsequent transformation into DH5a

Ligation of Ara_503m with pSB1A2_tctB162m_tphCm/pSB1C3_tctB162m_tphCm and subsequent transformation into DH5a. Screening the transformants using colony-PCR

only colony 1, 2 and 3 have the right insert

Miniprep (Fermentas Kit) and Nanodrop measurement of:

pSB1A2_Ara_tctA503m : 278.0 ng/µl 260/280= 1.86

pSB1C3_Ara_tctA503m : 169.0 ng/µl 260/280= 1.87

pSB1C3_Ara_1 : 59.0 ng/µl 260/280= 1.90

pSB1C3_Ara_4 : 55.5 ng/µl 260/280= 1.80

pSB1C3_Ara_tctA503m_tctB162m_tphCm : 188.8 ng/µl 260/280= 1.86

pSB1C3_Ara_tctA503m_tctB162m_tphCm :168.8 ng/µl 260/280= 1.84

pSB1C3_Ara_tctA503m_tctB162m_tphCm :271.3 ng/µl 260/280= 1.85

pSB1C3_Ara_XylE : 149.8 ng/µl 260/280= 1.86

pSB1C3_tctB197_tphC : 95.8 ng/µl 260/280= 1.84

pSB1A2_tctB197_tphC : 69.0 ng/µl 260/280= 1.88

Sequencing of 3 different pSB1C3_Ara_tctA503m_tctB162m_tphC

Analysis of mutagenic PCR products of 505_8_2 and restriction digest with PstI

Restriction digest of Ara_RBS with EcoRI and SpeI and of pSB1C3_505m with EcoRI and XbaI. Building pSB1C3_Ara_505m by ligation and subsequent transformation into DH5a.

week 13 (13.-17.08.12)

Restriction digest of pSB1C3_197_tphCm with EcoRI and XbaI

Checking the transformation of DH5a with pSB1C3_Ara_505m by using colony-PCR

Sequencing pSB1C3_Ara_XylE, pSB1C3_Ara_tctA503m_tctB162m_tphCm (=Operon1), pSB1C3_505m

Restriction digest of Ara_RBS with EcoRI and SpeI and of pSB1C3_tphCm with EcoRI and XbaI. Building pSB1C3_Ara_tphCm by ligation and subsequent transformation into DH5a

Restriction digest of pSB1C3_Ara_tctA505m with EcoRI and SpeI. Ligation of Ara_tctA505m and tctB197_tphCm into pSB1C3

week 14 (20.-24.08.12)

Checking the transformation of DH5a with pSB1C3_Ara_tphCm using colony-PCR

week 15 (27.-31.08.12)

Restriction digest of pSB1C3_Ara_tphCm and pSB1C3_Ara_tctA505m with PstI and SpeI

Restriction digest of pSB1C3_197_tphCm with XbaI and PstI.

Building the construct pSB1C3_Ara_tctA505m_197_tphCm (= Operon2) by ligation. Subsequent transformation into DH5a

Checking the transformation of DH5a with Operon2 using colony-PCR

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 <span style="font-size:188%;"><span style="color:#00689D;">Transport</span></span>
 ===Week 1 (21.-25.05.12)===
 * First [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony PCR] of ''Comamonas testosteroni'' for isolation of the following genes: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tctB 197aa] (2), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 tctA_503aa] (3), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tctB 162aa] (4), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5). (Primer: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503 tctA_503 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick],  [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162 tctB_162 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB197 tctB_197 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick]). Analysis of [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] products by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]
 :https://static.igem.org/mediawiki/2012/1/15/W1_1_kolo_pcr.png
 * PCR product purification using the Promega-Kit
 * Nanodrop measurements of the purified PCR products:
 :(1) : 19.5 ng/µl 260/280=1.58
 :(2) : 61.9 ng/µl 260/280=1.75
 :(3) : 75.1 ng/µl 260/280=1.75
 :(4) : 93.3 ng/µl 260/280=1.85
 :(5) : 91.3 ng/µl 260/280=1.58
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of PCR products by SpeI and EcoRI, heat inactivation after digestion
 * Analysis of restriction digest by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]
 :https://static.igem.org/mediawiki/2012/1/15/w1_2_restriktionsverdau_s8.png
 * Purification of the bands gained form gel electrophoresis (Promega-Kit)
 * Nanodrop measurements of the purified products:
 :(1) : 8.3 ng/µl 260/280=1.94
 :(2) : 8.9 ng/µl 260/280=1.80
 :(3) : 13.0 ng/µl 260/280=1.57
 :(4) : 1.5 ng/µl 260/280=3.08
 :(5) : 3.7 ng/µl 260/280=2.26
 ===week 2 (28.05.-01.06.12)===
 * Purified products from 1. week were [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation ligated] into pSB1A2 and subsequently [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformed] into DH5a (in spite of low concentration)
 * Overnight incubation on LB agar plates; no growth detectable
 * Second approach to isolate the five genes from ''Comamonas t.'' using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] (Primer: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503 tctA_503 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick],  [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162 tctB_162 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB197 tctB_197 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick])
 * Analysis of PCR products by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]; without results; modification of the PCR protocols: adding DMSO
 * Third approach to isolate the five genes from ''Comamonas t.'' by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] (Primer: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503 tctA_503 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick],  [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162 tctB_162 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB197 tctB_197 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick])
 * PCR product purification using the Promega-Kit: ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tctB 197aa] (2), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 tctA_503aa] (3), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tctB 162aa] (4), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5))
 :https://static.igem.org/mediawiki/2012/f/fc/W2_1_kolo_pcr_2_s19.png
 :(1): no PCR-product
 * Nanodrop measurements of the purified PCR products:
 :(2) : 56.1 ng/µl 260/280=1.87
 :(3) : 66.8 ng/µl 260/280=1.92
 :(4) : 72.8 ng/µl 260/280=1.87
 :(5) : 68.8 ng/µl 260/280=1.82
 ===week 3 (04.-08.06.12)===
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of the purified PCR products and of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (leftover of the 1. week) by SpeI and EcoRI
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of pSB1A2 using SpeI and EcoRI
 * Dephosphorylation of the restriction reactions by using antarctic Phosphatase
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of the five genes into pSB1A2 and subsequently [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] into DH5a (incubation at 37°C)
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1), 2 approaches (Primer:[https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick])
 * Analysis of PCR products by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]. Only one of them showed the expected band. The band was excluded and purified via the Promega-Kit.
 :https://static.igem.org/mediawiki/2012/1/11/W3_1_kolo_pcr_505_s23.png
 * Nanodrop measurement of the purified product:
 :(1.1) =8.9 ng/µl 260/280=2,22
 :Because of low concentration and contamination the PCR was repeated and the product was purified again by the Promega-Kit.
 * Performing a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR]-screen of the transformed cells using the Taq-Polymerase. Verification of 4 colonies per plate. (Primer: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503 tctA_503 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick],  [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162 tctB_162 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB197 tctB_197 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick])
 :PCR did not work
 * Testing the Taq-Polymerase for function. The Phu-Polymerase was used for comparison. Only the approach using the Phu-Polymerase showed the expected bands in [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis].
 :https://static.igem.org/mediawiki/2012/5/5a/W3_2_taq-phu_s27.2.png
 * Screening was repeated with Phu-Polymerase; no bands visible
 ===week 4 (11.-15-06.12)===
 * Inoculation of LB-media with DH5a_pSB1A2 and DH5a_pSB1C3; overnight incubation at 37°C
 * Miniprep of DH5a_pSB1A2 and DH5a_pSB1C3 using the Promega-Kit
 * Nanodrop measurements of the preparation:
 :pSB1A2 : 136.1 ng/µl 260/280= 1.93
 :pSB1C3 : 265.1 ng/µl 260/280= 1.89
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of pSB1A2 and pSB1C3 with EcoRI and SpeI. Afterwards dephosphorylation by using Antarctic phosphatase.
 * Analysis of the restriction products through [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]; for comparison the uncropped vectors were also analyzed.
 :https://static.igem.org/mediawiki/2012/6/69/W4_1_restriktion_der_vektoren.png
 :pSB1C3 shows several bands (Insert, vector without insert, linearized vector with insert); concentration of pSB1A2 is very low, only one band visible
 * Approach to isolate the following genes from ''Comamonas t.'' by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR]: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tctB 197aa] (2), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 tctA_503aa] (3), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tctB 162aa] (4) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5) by [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] (Primer: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503 tctA_503 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick],  [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162 tctB_162 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB197 tctB_197 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick])
 * Analysis of PCR products by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]
 :https://static.igem.org/mediawiki/2012/9/9f/W4_2_koloPCRcoma_197-503-162.png
 :Isolation of (1) and (5) did not work
 * PCR product purification by using the Promega-Kit
 * Nanodrop measurements of the purified PCR products:
 :(2) : 18.3 ng/µl 260/280=2.03
 :(3) : 21.2 ng/µl 260/280=1.95
 :(4) : 23.9 ng/µl 260/280=2.05
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of purified PCR products (2), (3) and (4) using SpeI and EcoRI
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of the digested genes ((2), (3), (4)) into pSB1A2 and pSB1C3
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation Transformation] of DH5a with pSB1A2_197, pSB1A2_503, pSB1A2_162, pSB1C3_197, pSB1C3_503, pSB1C3_162 (6 different transformation approaches). Overnight incubation at 37°C
 * Screening of the transformants by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] (Primer: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503 tctA_503 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick],  [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162 tctB_162 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB197 tctB_197 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick]). Verification of 6 colonies for every transformation. Afterwards the PCR products were analyzed by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]
 :https://static.igem.org/mediawiki/2012/d/dd/W4_3_koloPCRscreen1.png
 :https://static.igem.org/mediawiki/2012/4/40/W4_4_koloPCRscreen2.png
 :positive colonies are marked
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR Colony-PCR] of ''Comamonas t.'' to isolate [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1) und [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5) (Primer: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick])
 * Analysis of PCR products (Colony-PCR) and the ligation approach by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]
 :https://static.igem.org/mediawiki/2012/f/f7/W4_5_ligation%2BkoloPCR505-322.png
 :PCR of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] shows only the band of the undesired smaller product
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5) and subsequent [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] into pSB1A2 and pSB1C3
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation Transformation] of DH5a with pSB1A2_322 und pSB1C3_322. [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] (Primer: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick]) used to check for successful transformation<!-- bild fehlt!! -->
 * Isolation of ''Comamonas t.'' genome, because it seems impossible to amplificate [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1) from a fluid ''Comamonas t.'' culture.
 ===week 5 (18.-22.06.12)===
 * Introduction of a nomenclature: A/C gene/colony: pSB1A2 = A; pSB1C3 = C; gene: tctA 505aa = 1, tctB 197aa = 2, tctA 503aa = 3, tctB 162aa = 4, tphC 322aa = 5; colony = 1...X
 * Producing fluid cultures of the positive transformants
 * Miniprep of the fluid culures by using the Frementas-Kit. Afterwards Nanodrop measurements of the preparations
 :https://static.igem.org/mediawiki/2012/8/8f/W5_1_positive_transformanden.png
 :A2.3 : 125.9 ng/µl 260/280= 1.62
 :A3.1 : 103.3 ng/µl 260/280= 1.72
 :A4.6 : 147.2 ng/µl 260/280= 1.70
 :A4.5 : 108.7 ng/µl 260/280= 1.86
 :A5.12 : 88.0 ng/µl 260/280= 1.87
 :A5.8 : 85.01 ng/µl 260/280= 1.78
 :A3.2 : 87.3 ng/µl 260/280= 1.81
 :C4.6 : 70.5 ng/µl 260/280= 1.81
 :C4.1 : 41.2 ng/µl 260/280= 1.85
 :C4.4 : 104.2 ng/µl 260/280= 1.85
 :C2.1 : 132.2 ng/µl 260/280= 1.85
 :C2.6 : 188.1 ng/µl 260/280= 1.75
 * [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] using a rest of purified [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1) (Primer: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick]), subsequent [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]
 * Another [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] using the transformants (2)-(5) as template (Primers: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503 tctA_503 Biobrick],  [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162 tctB_162 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB197 tctB_197 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick]); analyzing the PCR product by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]
 :https://static.igem.org/mediawiki/2012/1/15/W5_2_screen.png
 :3 positive transformants were picked for inoculation of fluid media (A34, A43; C513)
 * Miniprep (Fermentas Kit) of the fluid culures and Nanodrop measurements:
 :A2.4 : 202,1 ng/µl 260/280= 1,63
 :A2.5 : 126,5 ng/µl 260/280= 1,90
 :A2.6 : 107,5 ng/µl 260/280= 1,81
 :A4.2 : 133,8 ng/µl 260/280= 1,59
 :C2.3 : 187,8 ng/µl 260/280= 1,87
 :C2.4 : 270,7 ng/µl 260/280= 1,80
 :C4.1 : 103,8 ng/µl 260/280= 1,80
 * The Miniprep products were [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digested] by restriction enzymes, each was cut once with EcoRI and twice with EcoRI/SpeI. Analisys of the digested products by [[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]
 :https://static.igem.org/mediawiki/2012/4/4b/W5_3_miniprep1x2x.png
 ===week 6 (25.-29.06.12)===
 * Further [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] of ''Comamonas t.'' (3 approaches) and [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] with genomic ''Comamonas t.'' DNA as template to obtain [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1), 2 approaches each (Primer: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick]). Subsequent analisys of the PCR products by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]. All PCR of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] show only the band of the undesired smaller product.
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] off the digested genes into pSB1A2 and [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] into DH5a. Overnight incubation at 37°C
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR Colony-PCR] of ''Comamonas t.'' and [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] with genomic ''Comamonas t.'' DNA as template to obtain [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1). [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tctB 197aa] (2), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 tctA_503aa] (3), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tctB 162aa] (4), [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 tphC 322aa] (5) used as control (Primers:  [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503 tctA_503 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick],  [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162 tctB_162 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB197 tctB_197 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick])
 :https://static.igem.org/mediawiki/2012/c/ce/W6_1_PCR_505.png
 * Miniprep and Nanodrop measurement of:
 :A3.1 : 111.6 ng/µl 260/280= 1.72
 :A3.4 : 79.5 ng/µl 260/280= 1.66
 :C4.6 : 69.6 ng/µl 260/280= 1.76
 :A4.2 : 96.9 ng/µl 260/280= 1.62
 :A2.4 : 109.7 ng/µl 260/280= 1.72
 :A2.5 : 160.8 ng/µl 260/280= 1.79
 :A5.13 : 188.7 ng/µl 260/280= 1.85
 :A5.12 : 114.5 ng/µl 260/280= 1.81
 * Checking the Miniprep with [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] (Primers: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503 tctA_503 Biobrick],  [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162 tctB_162 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB197 tctB_197 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick]) and [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]
 :https://static.igem.org/mediawiki/2012/7/7c/W6_2_miniprep.png
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR Colony-PCR]-Screen of the transformants (Primer: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick]) and analisys of the PCR products by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]
 :No bands visible
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] into pSB1C3 and subsequently [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] into DH5a. No colonies
 *  The following vectors were sequenced: pSB1A2_197.4, pSB1A2_197.5, psSB1A2_503.1, pSB1A2_503.4, pSB1A2_162.2, pSB1C3_162.6, pSB1A2_322.12, pSB1A2_322.13
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] using EcoRI/SpeI. [[Ligation]] into  pSB1C3 and subsequently [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] into DH5a.
 * Preperations for pure culture: [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] of A2.4, A2.5, A3.1, A3.4, A4.2, C4.6, A5.12, A5.13 (2 colonies each, Primers: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503 tctA_503 Biobrick],  [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162 tctB_162 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB197 tctB_197 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick]) and afterwards checking the PCR products by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]. Positive colonies were picked, streaked onto fresh media and incubated again.
 * As A5.12 and A5.13 are missing the right insert the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] was repeated (5 and 6 colonies were used, Primer: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick]). To provide additional monitoring the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] was repeated for A2.5_2, A3.1_2 und A4.2_2. 2 positive colonies were again streaked onto fresh media.
 * The Arabinose-Promotor ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara]) was isolated from pbad_turbo_gfp and colonies containing this plasmid (3 approaches each). Approach  T2 is further in use.
 :https://static.igem.org/mediawiki/2012/f/fd/W6_3_ara.png
 ===week 7 (02.-06.07.12)===
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] and pXylE using EcoRI and SpeI. [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of the restricted products and [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] into  DH5a. No colonies
 :https://static.igem.org/mediawiki/2012/0/00/W7_1_ara_restriktion.png
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of pSB1A2 using EcoRI and SpeI
 * DH5a-cells containing the vectors A4.2_2, A3.1_2, A2.5_2, A5.13_5 were diluted in glycerol (20%) and freezed (-80°C).
-* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/mutagenic_PCR Mutagenic PCR] of A4.2, A5.13, A3.1 to eliminate the PstI restriction site (Primers: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503#tctA503_Pst1 tctA_503_PstI], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162#tctB162_Pst1 tctB_162_PstI], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC#tphC_Pst1 tphC_PstI])
-* [**********]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/mutagenic_PCR Mutagenic PCR] of A4.2, A5.13, A3.1 to eliminate the PstI restriction site (Primers: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503#tctA503_Pst1 tctA_503_PstI], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162#tctB162_Pst1 tctB_162_PstI], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC#tphC_Pst1 tphC_PstI]). [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation Transformation] into DH5a
 * Checking the DH5a-cells for correct transformation with the vectors after the mutagenic PCR by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] (checking 7 colonies each, Primers: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA503 tctA_503 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctB162 tctB_162 Biobrick], [https://2012.igem.org/Team:TU_Darmstadt/Materials/tphC tphC Biobrick]). The PCR products were [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest digested] with PstI. The gel was alternately loaded with digested and uncut PCR products.
 :https://static.igem.org/mediawiki/2012/b/b7/W7_2_trafo_mit_mut.png
 * The transformation of DH5a with pSB1C3_tctA_505aa was verified by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] (Primers: [https://2012.igem.org/Team:TU_Darmstadt/Materials/tctA505 tctA_505 Biobrick]). The positive colonies were used for Miniprep
 * Miniprep (Fermentas Kit) and Nanodrop measurement:
 :A5.13m_1 : 105.2 ng/µl 260/280= 1.84
 :A5.13m_2 : 223.0 ng/µl 260/280= 1.87
 :A5.13m_3 : 110.6 ng/µl 260/280= 1.87
 :A5.13m_4 : 99.6 ng/µl 260/280= 1.90
 :A5.13m_6 : 326.6 ng/µl 260/280= 1.88
 :A5.13m_7 : 145.0 ng/µl 260/280= 1.84
 :A3.1m_1 : 241.7 ng/µl 260/280= 1.87
 :A3.1m_2 : 188.4 ng/µl 260/280= 1.89
 :A3.1m_3 : 112.4 ng/µl 260/280= 1.89
 :A3.1m_4 : 173.1 ng/µl 260/280= 1.85
 :A3.1m_5 : 111.6 ng/µl 260/280= 1.85
 :A3.1m_6 : 138.1 ng/µl 260/280= 1.87
 :A3.1m_7 : 178.9 ng/µl 260/280= 1.86
 :A4.2m_1 : 57.0 ng/µl 260/280= 1.84
 :A4.2m_2 : 46.5 ng/µl 260/280= 1.77
 :A4.2m_3 : 55.7 ng/µl 260/280= 1.88
 :A4.2m_4 : 111.3 ng/µl 260/280= 1.82
 :A4.2m_5 : 236.9 ng/µl 260/280= 1.89
 :A4.2m_6 : 69.3 ng/µl 260/280= 1.89
 :A4.2m_7 : 72.3 ng/µl 260/280= 1.85
 :C1.2 : 132.9 ng/µl 260/280= 1.72
 * new [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] of DH5a with pXylE_Ara
 ===week 8 (09.-13.07.12)===
 * Checking the transformation of DH5a with pSB1C3_tctA_505aa again ([https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR]) [*]. The colonies Nr. 2, 8 and 10 were picked and streaked onto fresh media.
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] of DH5a_pXylE_Ara [*]
 :No positive colonies
 * Sequencing the following vectors: pSB1A2_4.2m_5, pSB1A2_4.2m_4, pSB1A2_5.13m_3, pSB1A2_5.13m_2, pSB1A2_3.1m_1, pSB1A2_3.1m_5, pSB1A2_3.1m_7
 * Checking the transformation of DH5a with A1 (5 colonies) and C1 (7 colonies) by using Colony-PCR [?]
 * Checking the transformation of DH5a with pSB1C3_tctA_505aa again by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR]. Only the colonies Nr. 2, 8 and 10 (see above) were checked (2 colonies each).
 :https://static.igem.org/mediawiki/2012/f/f0/W8_3_kolo_pcr_505.png
 * Another [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] of DH5a_pXy19_Ara
 * Miniprep (Fermentas Kit) and Nanodrop measurement of:
 :A5.13m_2 : 191.2 ng/µl 260/280= 1.84
 :A5.13m_3 : 168.1 ng/µl 260/280= 1.78
 :A2.5 : 193.0 ng/µl 260/280= 1.83
 :A3.1m_5 : 146.1 ng/µl 260/280= 1.82
 :A3.1m_1 : 250.1 ng/µl 260/280= 1.84
 :A4.2m_4 : 124.8 ng/µl 260/280= 1.85
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of A5.13m_2, A5.13m_3, A3.1m_1, A3.1m_5, A2.5, A4.2m_4 with EcoRI and SpeI. The restriction was done to enable the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation ligation] of the mutated genes into pSB1C3. Afterwards DH5a was [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformed] with the ligation product.
 * Miniprep (Fermentas Kit) and Nanodrop measurement of:
 :C505_2_2 : 253.6 ng/µl 260/280= 1.60
 :C505_2_3 : 274.7 ng/µl 260/280= 1.88
 :C505_8_2 : 462.1 ng/µl 260/280= 1.38
 :C505_8_3 : 149.1 ng/µl 260/280= 1.84
 :C505_10_3 : 160.4 ng/µl 260/280= 1.98
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of the Miniprep products with EcoRI and EcoRI/SpeI. Afterwards the restriction products were analyzed by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]. Gel loading: uncut, cut once, cut twice
 :https://static.igem.org/mediawiki/2012/7/7e/W8_4_restriktion_505.png
 :C505_2_3 und C505_8_2 were selected for mutagenic PCR.
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of pSB1C3 with EcoRI and SpeI
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of pXylE with EcoRI and XbaI and of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] with EcoRI and SpeI. Dephosphorylation of pXylE. [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of pXylE and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara]
 ===week 9 (16.-20.07.12)===
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/mutagenic_PCR mutagenic PCR] of C505_2_3 and C505_8_2. (Did not work)
 * [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 tct_B162m] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 tct_B197m] with primer to add a RBS in front and behind the gene
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of the PCR products with EcoRI and SpeI for [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation ligation] into pSB1C3_5.13m (tphC)
 * Adding a RBS to one site of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] using PCR with the reverse primer
 * Miniprep (Fermentas Kit) and Nanodrop measurement of:
 :C3.1m_1 : 278.9 ng/µl 260/280= 1.88
 :C4.2m_4 : 214.2 ng/µl 260/280= 1.86
 :5.13m_3 : 256.3 ng/µl 260/280= 1.88
 :C2.5_2 : 44.8 ng/µl 260/280= 2.00
 :C5.13m_2 : 60.8 ng/µl 260/280= 1.89
 ===week 10 (23.-27.07.12)===
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of pSB1C3_5.13m_3_2 and pSB1C3_5.13m_3_3 with EcoRI and XbaI. Afterwards the products are [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation ligated] with RBS_tctB162m_RBS and RBS_tctB197_RBS each.
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of A3.1m_1 for [[ligation]] with Ara
 *  [[Digestion]] of pXylE for [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation ligation] with Ara_RBS
 * [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] of Ara_RBS and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] for [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation ligation] into pXylE and pSB1C3. Afterwards [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest restriction digest] with restriction enzymes: Ara_RBS is cut with with EcoRI and SpeI and Ara is cut with EcoRI and XbaI
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of the restricted PCR-products to:  pSB1A2_tctB197_tphCm, pSB1C3_tctB162m_tphCm, pSB1C3_tct197_tphCm, pSB1A2_tctB162m_tphCm, pSB1A2_Ara_tctA503m, pSB1C3_Ara_tctA503m, pSB1A2_Ara_XylE and [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] into  DH5a
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/mutagenic_PCR mutagenic PCR] of 505_8_2
 ===week 11 (30.07.-03.08.12)===
 * Checking [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] of DH5a by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR]: pSB1A2_tctB197_tphCm, pSB1C3_tctB162m_tphCm, pSB1C3_tct197_tphCm, pSB1A2_tctB162m_tphCm, pSB1A2_Ara_tctA503m, pSB1C3_Ara_tctA503m, pSB1A2_Ara_XylE. Positive colonies are picked and used for inoculation of fluid media.
 * Miniprep (Fermentas Kit) of fluid culture and Nanodrop measurement of:
 :pSB1C3_503 : 110.0 ng/µl 260/280= 1.82
 :pSB1A2_tctB197_tphCm : 32.0 ng/µl 260/280= 2.10
 :pSB1A2_Ara_tctA503m : 202.0 ng/µl 260/280= 1.78
 :pSB1A2_tctB162m_tphCm : 36.5  ng/µl 260/280= 1.91
 :pSB1C3_Ara_tctA503m : 133.2 ng/µl 260/280= 1.83
 :pSB1C3_tctB162m_tphCm : 35.2 ng/µl 260/280= 1.93
 :pSB1C3_tct197_tphC : 30.6 ng/µl 260/280= 1.88
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of pSB1A2_Ara_tctA503m, pSB1C3_Ara_tctA503m, pSB1A2_tctB162m_tphCm and pSB1C3_tctB162m_tphCm and building the final constructs by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation ligation]: pSB1A2_tctA503m_tctB162m_tphCm und pSB1C3_tctA503m_tctB162m_tphCm. [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation Transformation] of the final constructs into DH5a.
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation Transformation] of pSB1C3_503 into DH5a
 *  Checking the mutagenic PCR product of 505_8_2 and the transformation of DH5a with pSB1C3_503 by [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR]
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] und C4.2_4_1 using EcoRI and PstI, building pSB1C3_Ara by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation ligation] and [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] of the vector into DH5a
 * Miniprep (Fermentas Kit) and Nanodrop measurement of:
 :pSB1C3_Ara : 86.7 ng/µl 260/280= 1.83
 :pSB1C3_505m : 113.8 ng/µl 260/280= 1.85
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of pSB1C3_505m with PstI. Checking if the restriction site has been successfully removed
 :[[File:W11_1_rest_mut_505.jpg|100px]]
 :restriction site hasn't been removed
 * Screnning for transformation of DH5a with pSB1A2_tctA503m_tctB162m_tphCm and pSB1C3_tctA503m_tctB162m_tphCm using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR] [*]
 * Checking the constructs pSB1A2/pSB1C3_Ara_503m and pSB1A2/pSB1C3_tctB162m_tphCm by [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] [*]
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] into pSB1C3
 * Checking if pSB1A2_tctB162m_tphCm and pSB1C3_tctB162m_tphCm have the right Prefix/Suffix by digesting both with EcoRI and XbaI and with EcoRI and PstI [*]
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of pSB1A2_tctB162m_tphCm and pSB1C3_tctB162m_tphCm with Ara_503
 *  Miniprep (Fermentas Kit) and Nanodrop measurement of:
 :pSB1A2_tctB162m_tphCm_1 : 45.3 ng/µl 260/280= 1.90
 :pSB1A2_tctB162m_tphCm_2 : 74.5 ng/µl 260/280= 1.82
 :pSB1C3_tctB162m_tphCm_1 : 95.8 ng/µl 260/280= 1.86
 :pSB1C3_tctB162m_tphCm_2 : 85.7 ng/µl 260/280= 1.81
 :pSB1C3_tctB162m_tphCm_3 : 100.1 ng/µl 260/280= 1.84
 :pSB1C3_tctB162m_tphCm_4 : 79.9 ng/µl 260/280= 1.88
 :pSB1C3_tctB162m_tphCm_5 : 89.8 ng/µl 260/280= 1.85
 * Sequencing pSB1A2_tctB162m_tphCm_2 and pSB1C3_tctB162m_tphCm_5
 ===week 12 (06.-10.08.12)===
 * Miniprep (Fermentas Kit) and Nanodrop measurement of:
 :pSB1A2_tctB162m_tphCm_2 : 55.7 ng/µl 260/280= 1.81
 :pSB1C3_tctB162m_tphCm_5 : 101.2 ng/µl 260/280= 1.92
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/mutagenic_PCR Mutagenic PCR] of pSB1C3_505_8_2 and subsequent [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] into DH5a
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of Ara_503m with pSB1A2_tctB162m_tphCm/pSB1C3_tctB162m_tphCm and subsequent [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] into DH5a. Screening the transformants using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR]
 :[[File:W12_1_kolo_operon1.JPG|250px]]
 :only colony 1, 2 and 3 have the right insert
 * Miniprep (Fermentas Kit) and Nanodrop measurement of:
 :pSB1A2_Ara_tctA503m : 278.0 ng/µl 260/280= 1.86
 :pSB1C3_Ara_tctA503m : 169.0 ng/µl 260/280= 1.87
 :pSB1C3_Ara_1 : 59.0 ng/µl 260/280= 1.90
 :pSB1C3_Ara_4 : 55.5 ng/µl 260/280= 1.80
 :pSB1C3_Ara_tctA503m_tctB162m_tphCm : 188.8 ng/µl 260/280= 1.86
 :pSB1C3_Ara_tctA503m_tctB162m_tphCm :168.8  ng/µl 260/280= 1.84
 :pSB1C3_Ara_tctA503m_tctB162m_tphCm :271.3  ng/µl 260/280= 1.85
 :pSB1C3_Ara_XylE : 149.8 ng/µl 260/280= 1.86
 :pSB1C3_tctB197_tphC : 95.8 ng/µl 260/280= 1.84
 :pSB1A2_tctB197_tphC : 69.0 ng/µl 260/280= 1.88
 * Sequencing of 3 different pSB1C3_Ara_tctA503m_tctB162m_tphC
 * Analysis of mutagenic PCR products of 505_8_2 and [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest restriction digest] with PstI
 :[[File:W12_2_505m_restriktion.JPG|400px]]
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of Ara_RBS with EcoRI and SpeI and of pSB1C3_505m with EcoRI and XbaI. Building pSB1C3_Ara_505m by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation ligation] and subsequent [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] into DH5a.
 ===week 13 (13.-17.08.12)===
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of pSB1C3_197_tphCm with EcoRI and XbaI
 * Checking the transformation of DH5a with pSB1C3_Ara_505m by using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR]
 :[[File:W13_1_505_mut_PCR_von_Basti.JPG|500px]]
 * Sequencing pSB1C3_Ara_XylE, pSB1C3_Ara_tctA503m_tctB162m_tphCm (=Operon1), pSB1C3_505m
 *[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of Ara_RBS with EcoRI and SpeI and of pSB1C3_tphCm with EcoRI and XbaI. Building pSB1C3_Ara_tphCm by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation ligation] and subsequent [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] into DH5a
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of pSB1C3_Ara_tctA505m with EcoRI and SpeI. [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of Ara_tctA505m and tctB197_tphCm into pSB1C3
 ===week 14 (20.-24.08.12)===
 * Checking the transformation of DH5a with pSB1C3_Ara_tphCm using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR]
 :[[File:W14_1_kolo.JPG|250px]]
 ===week 15 (27.-31.08.12)===
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of pSB1C3_Ara_tphCm and pSB1C3_Ara_tctA505m with PstI and SpeI
 * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of pSB1C3_197_tphCm with XbaI and PstI.
 * Building the construct pSB1C3_Ara_tctA505m_197_tphCm (= Operon2) by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation ligation]. Subsequent [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation transformation] into DH5a
 * Checking the transformation of DH5a with Operon2 using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR colony-PCR]
 :[[File:W15_1_operon2_Kolo.jpg|100px]]