Team:TU Darmstadt/Labjournal/Transport

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

• 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 electrohporesis

• 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))

(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.

• 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.

• 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

• Restriction Restriction digest of pSB1A2 and pSB1C3 with EcoRI and SpeI. Afterwards dephosphorylation by using Antarctic phosphatase.
• Analysis of the restriction products through agarose gel electrohporesis; 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]
• Analysis of PCR products by agarose gel electrohporesis

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

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

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

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

• 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 electrohporesis

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.