Team:TU Darmstadt/Labjournal/Transport

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Transport

Contents

Overview

Klonierung.png

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). (Primers: 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
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
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 ligated into pSB1A2 and subsequently transformed into DH5a (in spite of low concentration)
  • Overnight incubation on LB agar plates; no growth detectable
  • 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))
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)

  • 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
  • Dephosphorylation of the restriction reactions by using antarctic Phosphatase
  • colony-PCR of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1), 2 approaches (Primers: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.
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.
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.
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
  • Restriction digest of pSB1A2 and pSB1C3 with EcoRI and SpeI. Afterwards dephosphorylation by using Antarctic phosphatase.
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 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] (Primers: tctA_503 Biobrick, tctA_505 Biobrick, tctB_162 Biobrick, tctB_197 Biobrick, tphC Biobrick)
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
  • 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
W4 3 koloPCRscreen1.png
W4 4 koloPCRscreen2.png
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) (Primers: tctA_505 Biobrick, tphC Biobrick)
W4 5 ligation+koloPCR505-322.png
PCR of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] shows only the band of the undesired smaller product
  • 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] (Primers: 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
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) (Primers: tctA_505 Biobrick), subsequent Agarose gel electrophoresis
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
W5 3 miniprep1x2x.png


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 (Primers: 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.
  • 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)
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
W6 2 miniprep.png
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
  • As A5.12 and A5.13 are missing the right insert the colony-PCR was repeated (5 and 6 colonies were used, Primers: 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.
W6 3 ara.png


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
W7 1 ara restriktion.png
  • 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).
W7 2 trafo mit mut.jpg
  • 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

week 8 (09.-13.07.12)

  • Checking the transformation of DH5a with pSB1C3_tctA_505aa again using colony-PCR (Primers: tctA_505 Biobrick) [*]. The colonies Nr. 2, 8 and 10 were picked and streaked onto fresh media.
  • colony-PCR of DH5a_pXylE_Ara (Primers: Ara_RBS, no positve colonies, data not shown)
  • 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) using colony-PCR (Primers: tctA_505 Biobrick, data not shown)
  • Checking the transformation of DH5a with pSB1C3_tctA_505aa again by colony-PCR (Primers: tctA_505 Biobrick). Only the colonies Nr. 2, 8 and 10 (see above) were checked (2 colonies each).
W8 3 kolo pcr 505.png
  • 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
W8_4_restriktion_505.png
C505_2_3 und C505_8_2 were selected for mutagenic PCR.
  • 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)

  • [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 (Primers: tctB_162_RBS, tctB_197_RBS)
  • Adding a RBS to one site of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Ara] using [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] (Primers: Ara_RBS)
  • 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.
  • [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 (Primers: Ara_RBS). 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


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 (Primers: [http://partsregistry.org/Part:BBa_G00100 VF2], [http://partsregistry.org/Part:BBa_G00101 VR]). 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.
  • 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] (Primers: tctA_505 Biobrick, tctA_503 Biobrick)
  • 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
W11 1 rest mut 505.jpg
restriction site hasn't been removed
  • Screnning for transformation of DH5a with pSB1A2_tctA503m_tctB162m_tphCm and pSB1C3_tctA503m_tctB162m_tphCm using colony-PCR (Primers: [http://partsregistry.org/Part:BBa_G00100 VF2], [http://partsregistry.org/Part:BBa_G00101 VR]) [*]
  • Checking the constructs pSB1A2/pSB1C3_Ara_503m and pSB1A2/pSB1C3_tctB162m_tphCm by [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] (Primers: [http://partsregistry.org/Part:BBa_G00100 VF2], [http://partsregistry.org/Part:BBa_G00101 VR]) [*]
  • 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
  • Ligation of Ara_503m with pSB1A2_tctB162m_tphCm/pSB1C3_tctB162m_tphCm and subsequent transformation into DH5a. Screening the transformants using colony-PCR (Primers: [http://partsregistry.org/Part:BBa_G00100 VF2], [http://partsregistry.org/Part:BBa_G00101 VR])
W12 1 kolo operon1.JPG
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
W12 2 505m restriktion.JPG

week 13 (13.-17.08.12)

  • Checking the transformation of DH5a with pSB1C3_Ara_505m by using colony-PCR (Primers: [http://partsregistry.org/Part:BBa_G00100 VF2], [http://partsregistry.org/Part:BBa_G00101 VR])
W13 1 505 mut PCR von Basti.JPG
  • Sequencing pSB1C3_Ara_XylE, pSB1C3_Ara_tctA503m_tctB162m_tphCm (=Operon1), pSB1C3_505m

week 14 (20.-24.08.12)

  • Checking the transformation of DH5a with pSB1C3_Ara_tphCm using colony-PCR (Primers: [http://partsregistry.org/Part:BBa_G00100 VF2], [http://partsregistry.org/Part:BBa_G00101 VR])
W14 1 kolo.JPG

week 15 (27.-31.08.12)

  • 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 (Primers: [http://partsregistry.org/Part:BBa_G00100 VF2], [http://partsregistry.org/Part:BBa_G00101 VR])
W15 1 operon2 Kolo.jpg