Team:TU Darmstadt/Labjournal/Transport

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Transport

Contents

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). Analysis of [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] products by agarose gel electrohporesis
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 DH5α (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
  • Analysis of PCR products by agarose gel electrohporesis; without results; modification of the PCR protocols: adding DMSO
  • Third approach to isolate the five genes from Comamonas t. by colony-PCR
  • 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
  • Restriction 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 DH5α (incubation at 37°C)
  • colony-PCR of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 tctA 505aa] (1), 2 approaches
  • Analysis of PCR products by agarose gel electrohporesis. 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.
  • Performing a colony-PCR-screen of the transformed cells using the Taq-Polymerase. Verification of 4 colonies per plate.
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 electrohporesis.
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 DH5α_pSB1A2 and DH5α_pSB1C3; overnight incubation at 37°C
  • Miniprep of DH5α_pSB1A2 and DH5α_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
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]
  • Analysis of PCR products by agarose gel electrohporesis
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
  • Restriction 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 DH5α 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. Verification of 6 colonies for every transformation. Afterwards the PCR products were analyzed by agarose gel electrohporesis
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)
  • Analysis of PCR products (Colony-PCR) and the ligation approach by agarose gel electrohporesis
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
  • Restriction 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 DH5α with pSB1A2_322 und pSB1C3_322. [http://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] 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), subsequent agarose gel electrohporesis
  • Another colony-PCR using the transformants (2)-(5) as template; analyzing the PCR product by agarose gel electrohporesis
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-PCRof 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. Subsequent analisys of the PCR products by agarose gel electrohporesis. 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 DH5α. 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
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 DH5α. 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 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 DH5α.
  • 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) and afterwards checking the PCR products by agarose gel electrohporesis. Positive colonies were picked, streaked onto fresh media and incubated again.
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