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5.1.1 Cloning strategy: Generation of pUB6-ABCA3-HA vector

In order to generate puB6-hABCA3-HA vector with hemagglutinin tag (HA-tag) fused

to C-terminus of the ABCA3 gene, puB6/lacZ vector and pJet1/hABCA3-HA vector were

transformed into E.coli DH5 competent cells. Plasmid-DNA of both was isolated, restricted

through KpnI and XhoI and separated by gel electrophoresis. The following restriction

products were extracted with the help of QIAquick Gel Extraction Kit and Millipore Ultrafree-

DA: a 5320 bp KpnI and xhoI fragment containing pUB6 vector and a 5150 bp KpnI and xhoI

in water. For ligation, vector and insert, in 3:1 ratio, were incubated with T4 ligase and

corresponding reaction buffer first for 60 min at room temperature and second for 10 min at

65 °C. Products of ligation reaction were multiplied by transformation into E.coli DH5

competent cells. Two negative controls, the first one without insert and the second one

without insert and T4 ligase, were generated. When no bacterial colonies have grown in

these negative controls, plasmid DNA of the positive clones was isolated and their sequence

was confirmed by conventional sequencing.

Fig.11: Vector map of pUB6/V5-His: Plasmid vector puB6-hABCA3-HA for expression of C-terminal fusions of ABCA3 with hemagglutinin tag (HA-tag) was produced by modification of pUB6/V5-His vector. Own illustration according to a model from Dr. Suncana Kern.

5.1.2 Site-directed point mutagenesis

Site-directed point mutagenesis was performed with the help of QuickChange Site-

directed Mutagenesis Kit (Stratagene, La Jolla, California, USA) as recommended by the

manufacturer. The following cycling parameters as shown in Table 10 were used for the

introduction of point mutations into pEYFP-ABCA3- and pUB6-ABCA3-vectors.

Table 10: Optimal conditions for PCR in this study. Annealing temperature for introduction of the mutation L101P into pEYFP- ABCA3- vector was 52°C.

Time Temperature Cycles 5 min 95 °C 1

30 sec. 95 °C 12 1 min 52 °C 12 20 min 68 °C 12 10 min 68 °C 1

Point-mutagenesis products were digested with DpnI at 37 °C for 1 hour. DpnI is an

endonuclease that specifically digests methylated and hemimethylated DNA, which is

exclusively present in parental and non-mutated DNA template from bacterial

strains (115).

This specific digestion permits selection of mutation-containing synthesized DNA and

prevents transformation of non-mutated DNA into E.coli DH5 . Point-mutagenesis products

were subjected to a 0.8 % agarose gel.

5.1.3 DNA-sequencing

DNA-sequencing was conducted by Metabion International AG (Martinsried,

Germany).

5.1.4 E.coli DH5 culture

DH5

strain were grown on LB-Agar or in LB-Bouillon at 37°C. Media were

supplemented with 30 mg/ml of kanamycin or 100 mg/ml of ampicillin when necessary. For

subsequent plasmid-DNA isolation, 5 ml of cells were grown in LB-Bouillon at 37 °C with

continuous shaking overnight.1 ml of the preculture was inoculated into 100 ml of LB-Bouillon

and was grown further to optical density OD 600: 0.5-0.6. 1 ml of this overnight culture

supplemented with 33 % of glycerin was stored as a stock solution at -80 °C.

5.1.5 Generation of competent E.coli DH5

was inoculated into 100 ml of LB-Bouillon and grown further to optical density OD 600: 0.5-

0.6. For further procedure, DH5 cells were incubated on ice. After 5 min of inoculation on

ice, cells were collected at 4 °C and 5000 xg for 10 min. The cell pellet was resuspended in

40 ml of ice cold Tfb I buffer (100 mM RbCl

, 50 mM MnCl

, 30 mM K-acetat, 10 mM CaCl

-

2H

O, 15% glycerin, pH 5) and incubated on ice for further 30 min. Cells were collected at

4 °C and 5000 xg for 10 min, the pellet was disolved in ice cold Tfb II buffer (10 mM MOPS,

10 mM RbCl

, 75 mM CaCl

-2H

O, 15 % glycerin, pH 6.8), was frozen in liquid nitrogen and

stored at -80 °C.

5.1.6 Transformation in E.coli DH5

First, 15 l of point-mutagenesis products were added to DH5 cells and kept on ice

for 30 min. Second, cells were heat-treated at 42 °C for 2 min and immediately cooled down

on ice for 2 min. Third, cells were inoculated into 900 l of LB medium and grown at 37 °C for

1 hour. Fourth, cells were collected at 5000 xg for 2 min. Cell pellet was dissolved in 100 l

of media and cells were grown on LB agar with supplemented antibiotics at 37 °C overnight.

One sample with not transformed DH5 cells was used as a negative control. When no

colonies were detectable in negative controls, plasmid-DNA of transformed DH5 cells was

isolated, restricted, subjected to 0.8 % agarose gel and sequence was confirmed by

conventional sequencing.

5.1.7 Plasmid-DNA isolation

After transformation of pEYFP-ABCA3 or pUB6-ABCA3-HA vectors into DH5 strain

an overnight culture of DH5 strain was prepared. DNA was isolated out of DH5 strain

culture either with the help of QIAprep Spin Miniprep Kit or with NucleoBond Xtra Midi EF

according to the protocol provided by the manufacturer.

5.1.8 Restriction

Restriction was performed with 500 ng of DNA, 1 U of enzyme/1 g of DNA and 10x

corresponding reaction buffer were used. Mixtures were incubated at 37 °C for a minimum of

three hours. Results were subjected to a 0.8 % agarose gel.