2. MATERIAL AND METHODS
2.5 Molecular biologic methods
2.1.8 Modification of DNA
2.1.8.1 Overview about DNA-modifying enzymes used for cloning:
Restriction enzymes
DNA restriction endonucleases (restriction enzymes) recognition site
BamHI Fermentas / NEB G*GATCC
BglII Fermentas / NEB A*GATCT
EcoRI Fermentas / NEB G*AATTC
KpnI Fermentas GGTAC*C
SacI Fermentas / NEB GAGCT*C
SpeI Fermentas / NEB A*CTAGT
XbaI Fermentas / NEB T*CTAGA
XhoI Fermentas / NEB C*TCGAG
Table 13: Restriction enzymes used during cloning.
The enzyme recognition site is indicated by an asterisk. Enzymes (10 U/µL] including 10x buffers were from Fermentas and NEB.
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homing endonucleases recognition site I-SceI Fermentas;
Homing enzyme
5'-TAGGGATAA*CAGGGTAAT-3' 3'-ATCCC*TATTGTCCCATTA-5’
Table 14: Homing restriction enzyme I‐SceI.
This special enzyme was used for the excision of the expression cassette from the vector pSAT4‐35SP.RNAi prior to the transfer into the plant binary expression vector pRCS2‐ocs‐nptII. This enzyme has a non‐palindromic
recognition sequence (enzyme recognition site indicated by an asterisk) and is a rare‐cutter. Enzyme and 10x buffer were from Fermentas.
AlkalinePhosphatase and T4-DNA Ligase
Shrimp alkaline phosphatase (SAP)
(1 U/µL) Fermentas dephosphorylation of enzyme-
digested vectors T4 DNA Ligase (5 or 3 U/µL) Fermentas or
Promega Ligation Table 15: Ligase and shrimp alkaline phosphatase used during cloning.
Enzymes and 10x buffers were from Fermentas and Promega.
2.1.8.2 Enzymatic digestion of DNA for cloning
Enzymatic digestion of DNA was done for two purposes. An analytical digestion was performed to check the presence of the subcloned gene as well as correctness of restriction sites introduced by PCR prior to sequencing and further cloning steps. A preparative digestion was then applied in order to excise the subcloned gene fragment from the subcloning vector for cloning into the expression vector.
Restriction enzymes have been used from either Fermentas or New England Biolabs (NEB) according to their protocols.
Analytical digestion approaches:
Digestions using one enzyme were performed in 10 µL of reaction volume, digestion using 2 enzymes in 20 µL of reaction volume. Analytical digestion using one enzyme were performed with 1.5 µL of miniprep DNA (pGEM-T or pSTBlue-1 clones), 1 µL of recommended 10x reaction buffer, 0.5 – 1 µL of enzyme (10 U/µL) and water up to final volume of 10 µL. Digestion with 2 enzymes were made with 8 µL of miniprep DNA and 0.5 to 1 µL of each enzyme, 2 µL of recommended 10x buffer and water up to a final volume of 20 µL. NEB enzymes required in addition 1 µL of BSA (see Table 16).
Incubation followed for 1 ½ to 2 hours at 37 °C in an incubator followed by separation in agarose gel.
Preparative digestion approaches:
The following DNA volumes or amounts were digested according to miniprep concentration: 15 µL of high concentrated pGEM-T or pSTBLue-1 miniprep DNA or 3.5 µg of low concentrated expression vector miniprep DNA were digested. Preparation of the subcloned insert was done with 15 µL of miniprep DNA of pGEM-T or pSTBlue-1 clone, 10 µL of recommended 10x buffer, 1 – 2 µL of enzyme (either one or two) and water up to a reaction volume of 100 µL. NEB enzymes required in addition 1 µL of BSA (see Table 16). Incubation followed for 3 hours at 37 °C in an incubator followed by separation in agarose gel and purification of digested DNA fragment from agarose gel.
Preparation of empty plant expression vectors p21GT, ppT8 and p20F eCaMV35S for cloning was done with approximately 3.5 µg miniprep DNA, 10 µL of recommended 10x buffer, 1 – 2 µL of enzyme (either one or two) and water up to a reaction volume of 100 µL followed by dephosphorylation and separation in agarose gel and purification of cut DNA fragment from gel or direct purification from enzymatic reaction mix.
Single digestion pGEM-T, pSTBlue-1 p21GT, ppT8, p20F eCaMV35S
analytical preparative analytical preparative vector DNA (~ 3.5 µg/100 µL)
10x buffer [100x BSA, NEB]
enzyme dH2O
1,5 µL 1 µL [1 µL]
0.5 µL up to 10 µL
15 µL 10 µL [1 µL]
1 – 2 µL up to 100
1,5 µL 1 µL [1 µL]
0.5 µL up to 10 µL
3.5 µg 10 µL [1 µL]
1 - 2 µL up to 100 Double digestion
analytical preparative analytical preparative vector DNA (~ 3.5 µg/100 µL)
10x buffer [100x BSA, NEB]
enzyme 1 enzyme 2 dH2O
8 µL 2 µL [1 µL]
0.5 – 1 µL 0.5 – 1 µL up to 20 µL
15 µL 10 µL [1 µL]
1 - 2 µL 1 - 2 µL up to 100
8 µL 2 µL [1 µL]
0.5 – 1 µL 0.5 – 1 µL up to 20 µL
3.5 µg 10 µL [1 µL]
1 - 2 µL 1 - 2 µL up to 100 Table 16: Overview about enzymatic digestions.
Single digestions of subcloning‐vectors pGEM‐T and pSTBlue‐1 were used for clone analysis and testing of different reaction conditions on expression vectors. Preparative digestions were done to excise a subcloned gene fragment as well as for preparation of expression vector for cloning.
63 2.1.8.3 Dephosphorylation of vector DNA
Dephosphorylation of plant expression vectors has been performed directly after preparative digestion using one single restriction enzyme or blunt-end cutter. Using Fermentas buffers allowed direct addition of 1.5 µL Fermentas shrimp alkaline phosphatase (SAP, 10 U/µL) into the enzymatic reaction mix. The restriction enzyme reaction mix was inactivated by heating at 70 °C for 15 minutes. Dephosphorylation reaction followed at 37 °C for 30 minutes and the reaction was stopped by incubation at 70 °C for 15 minutes.
2.1.8.4 Cloning and Ligation
Cloning of genes investigated in this work was performed in two steps: Subcloning and cloning into the plant expression vector. Vectors for subcloning were from pGEM-T, pGEM-T Easy from Promega and pSTBlue-1 from Novagen. In both cases blue-white screening has been performed using 14 µL of 1 M IPTG (Fermentas) and 40 µL of 20 mg/mL X-Gal (Fermentas) in DMSO (Sigma-Aldrich).
Subcloning:
Following kits were used for subcloning: “pGEM®-T and pGEM®-T Easy Vector Systems” from Promega and “Novagen Perfectly Blunt® Cloning Kits” from Novagen.
The pGEM-T vector contains a T-overhang at 3’-position and therefore allows the so- called TA-cloning. This kind of cloning had to be applied after PCR amplification using
“Expand High Fidelity PCR System” from Roche since this kit contains a mix of Tgo DNA Polymerase and Taq DNA Polymerase, of which the latter one produces an A- overhang at 5’-position. Usage of “KAPA HiFi Proofreading DNA Polymerase” from Peqlab allowed blunt-end-cloning into pSTBlue-1 from Novagen.
Subcloning into pGEM-T was performed according to the kit protocol. 1 µL of vector DNA (50 ng) was mixed with either 1 µL 10x ligation buffer or 5 µL of 2X Rapid Ligation Buffer from Promega, 1 µL of T4 DNA Ligase (Promega or Fermentas), 2 µL of insert DNA fragment and water up to 10 µL, followed by incubation at 14 °C on a thermoblock over night followed by chemical transformation of E. coli Top10 or, in case of 2x Rapid Ligation Buffer, for 5 – 6 hours before transformation.
Subcloning into pSTBlue-1 has been performed as described in the kit protocol. First step was end conversion: 1 µL of PCR product was added to 4 µL of nuclease-free
water and 5 µL of End Conversion Mix (Novagen). The reaction was incubated at 22 °C for 15 minutes followed by enzyme inactivation at 75 °C for 5 minutes and cooling on ice for 2 minutes.
For ligation, 1 µL of pSTBlue-1 vector (50 ng) and 1 µL of T4 DNA Ligase (Promega or Fermentas) were added to the end-conversion reaction and incubated at 14 °C on a thermoblock. Transformation of E. coli Top10 followed the next day by chemical transformation.
Cloning into plant binary expression vectors:
The normal vector to insert molar ratio applied was 3 to 1, since the yield of digested and purified expression vectors was generally low in comparison to high concentrated insert coming from pGEM-T or pSTBlue-1 vectors. 1 µL of 10x T4 DNA ligation buffer (Fermentas) and 1 µL of T4 DNA ligase (Fermentas, Promega) were mixed with 1 to 2 µL of high concentrated insert DNA and 6 to 7 µL of digested vector up to a final reaction volume of 10 µL. Incubation followed over night at 14 °C on a thermoblock.
Table 17 summarizes all ligation reactions.
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Subcloning into pGEM-T (Promega) Subcloning into pGEM-T by rapid ligation pGEM-T vector (50 ng)
insert
10x ligation buffer 1,2) T4 DNA ligase 1,2) water up to 10 µL
1 µL 3 µL 1 µL 1 µL 4 µL
pGEM-T vector (50 ng) insert
2x Rapid ligation buffer 2) T4 DNA ligase 1,2)
1 µL 3 µL 5 µL 1 µL
total volume 10 µL total volume 10 µL
incubation over night at 14 °C
chemical transformation the following day
incubation for 5 – 6 hours at room temperature
chemical transformation Subcloning into pSTBlue-1 (Novagen)
1) End Conversion: 2) Ligation:
PCR product nuclease-free water end-conversion mix
1 µL 4 µL 5 µL
End-conversion reaction pSTBlue-1 vector (50 ng) T4 DNA ligase
10 µL 1 µL 1 µL
total volume 10 µL total volume 12 µL
incubation at 22 °C for 15 minutes
incubation at 75 °C for 5 minutes
incubation on ice for 2 minutes
incubation at 22 °C for 15 minutes
chemical transformation
Cloning into expression vectors p21GT, ppT8 and p20F e CaMV35S vector (digested, dephosporylated)
insert gene (digested) 10x ligation buffer 1) T4 DNA ligase 1) (water up to 10 µL)
3.5 µg 2 - 3 µL 1 µL 1 µL (1 µL)
total volume 10 µL
incubation over night at 14 °C
chemical transformation the following day Table 17: Overview about ligation reactions.
1) Fermentas, 2) Promega