Team:TU Darmstadt/Labjournal/Transport

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Transport

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 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 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 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 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.
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

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] and agarose gel electrohporesis

Colony-PCR-Screen of the transformants and analisys of the PCR products by agarose gel electrohporesis

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 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.
As A5.12 and A5.13 are missing the right insert the colony-PCR was repeated (5 and 6 colonies were used). 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 DH5α. No colonies

Restriction digest of pSB1A2 using EcoRI and SpeI
DH5α-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
Checking the DH5α-cells for correct transformation with the vectors after the mutagenic PCR (checking 7 colonies each). The gel was alternately loaded with digested and uncut PCR products.

The transformation of DH5α with pSB1C3_tctA_505aa was verified by colony-PCR. 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 DH5α with pXylE_Ara

week 8 (09.-13.07.12)

Checking the transformation of DH5α with pSB1C3_tctA_505aa again (colony-PCR) [*]. The colonies Nr. 2, 8 and 10 were picked and streaked onto fresh media.
colony-PCR of DH5α_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 DH5α with A1 (5 colonies) and C1 (7 colonies) by using Colony-PCR [?]
Checking the transformation of DH5α 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 DH5α_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 DH5α 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 electrohporesis. 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 DH5α
mutagenic PCR of 505_8_2

@@ Line 77: / Line 77: @@
 ===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 restriction digest] of pSB1A2 using 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 DH5α (incubation at 37°C)