Team:TU Darmstadt/Labjournal/Transport

From 2012.igem.org

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====Results====
====Results====
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* we got the purified Genes from ''C. testosteroni'' but the yield after purification is to low for futher use
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* We got the purified Genes from ''C. testosteroni'' but the yield after purification is to low for futher use
===week 2 (28.05.-01.06.12)===
===week 2 (28.05.-01.06.12)===

Revision as of 20:31, 25 September 2012

Transport

Contents

Overview

This lab journal describes a isolation and characterisation of the putative terephtalate (TPA) uptake system from Comamonas testosteroni KF-1 (C. testosteroni) in Escherichia Coli (E.Coli). The putative terephtalate system consist of three different domain, two alpha-helical transmembrane protein (11-12 and 4-5 alpha-helical transmembrane protein ) and one periplasmic terephtalate binding protein. The strain was purchased from DMSZ-German (link auf die seite) Collection of Microorganism and Cell Cultures (DMSZ no. 14576(auch link)). We designed two putative TPA uptake operons and the intake of TPA was checked by photometry, gas chromatography-mass spectrometry (GC-MS) and energy dispersive X-ray spectroscopy (EDX).


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
A
successful isolation of the five genes form C. testosteroni. Marker: 1kb DNA ladder, NEB
  • 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

Results

  • We got the purified Genes from C. testosteroni but the yield after purification is to low for futher use

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))
A
(1): no PCR-product. Marker: 1kb DNA ladder, NEB
  • 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

Results

  • Finally we got the genes 2, 3, 4 and 5 in acceptable concentrations

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
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