Molecular Cloning pp4-1

The aim of cloning the PP4-1 cDNA into vectors is to produce PP4-1 tagged with fluorescence protein (EYFP). This protein fusion will be used for investigating subcellular localization of PP4-1.

The cDNA will be inserted into EYFP-1 and EYFP-2 vector, respectively. Both pCAT-EYFP-1 and pCAT-EYFP-2 vectors share the same nucleotide sequence with the exception that pCAT-EYFP-1 will ensure that the resulting fusion-protein will carry the EYFP tag on the N–

Terminus, whereas the pCAT-EYFP-2 will give the resulting fusion-protein an EYFP tag at the C–

terminus.

Template of cDNA was taken from PP4-1 in pGEMT-easy vector. This template is Arabidopsis thaliana “Ecotype : Wassilewskija (WS)”, that shares the same sequence of the “Ecotype : Colombia” supplied by Prof. Jose Serrano (CNB in Madrid, Spain). High fidelity PCR was used for amplification of pp4-1 cDNA. The cDNA was then divided into two parts and labelled as PP4-1_A

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and PP4-1_B for amplified cDNA that will be inserted into pCAT-EYFP-1 and pCAT-EYFP-2, respectively. The target band in gel was cut and extracted.

3.4.2.1 Subclone PP4-1_A into pCAT-EYFP-1 vector

We would like to subclone PP4-1 cDNA to pCAT-EYFP-1 cloning vector in order to produce fusion-protein that will carry the EYFP tag on the N–Terminus. PP4-1_A that already amplified was digested using NotI and SacII restriction enzyme. The vector, pCAT-EYFP-1, was digested using NotI and SacII restriction enzyme as well. Those enzymes will cut specific recognition sites in the vector in order to make it linear. Both PP4-1_A and pCAT-EYFP-1 have to be treated with same restriction enzymes that create compatible ends.

The PP4-1_A was ligated into cloning vector, linear pCAT-EYFP-1. Following the method of ligation, reagents for each ligation mixture was prepared. The ligation mixture was left at 4^oC overnight before it was used in transformation of bacterial cells (Escherichia coli JM109) and transferred to LB agar plate containing Ampicillin. Selection of bacteria with ampicillin resulted survival of cells that have successfully inserted by plasmid. Five colonies from this plate were selected and PCR colony was performed to check those colonies (Figure 3-40).

Figure 3-40. The result of gel electrophoresis of colony PCR PP4-1 into pCAT-EYFP-1 vector. The size of expected PCR product is about 1.0 kbp. Colony PCR of PCR pp4-1 with vector pCAT-EYFP-1 showed all positive colonies.

The Primers were EYFP-C-Terf as forward Primer and AK73r as reversed Primer.

According to the positive result from Figure 3-40, two colonies from PP4-1_A--pCAT-EYFP-1 plate (number 1 and number 3) were selected and cultivated in LB Broth overnight. Plasmid isolation was performed using the plasmid minipreps kit and the concentration was measured. This plasmid was sent for sequencing. Table 3-21 provides the detail of mixture plasmid and selection primers that were used. The sequencing result is shown in Appendix 3-4.

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Table 3-21. The premixture of PCR PP4-1_A--pCAT-EYFP-1 plasmid for sequencing.

Sample Plasmid Plasmid (µL)

Water

(µL) Primer (3 µL) Sample Sequencing Name PP4-1_A--pCAT-EYFP-1

number 1

2.5 9.5 EYFP-C-Terf PP4-1YFPM_1ECter_EYFP-C-TERf 2.5 9.5 Ak92r PP4-1YFPM_1AK92r_AK92r PP4-1_A--pCAT-EYFP-1

number 3

1.0 11.0 EYFP-C-Terf PP4-1YFPM_3ECter_EYFP-C-TERf 1.0 11.0 Ak92r PP4-1YFPM_3AK92r_AK92r

According to the result in Appendix 3-4, all sequences clones of PP4-1_A--pCAT-EYFP-1 are correct can be used for subcellular localization. However, the clone PP4-1_A--pCAT-EYFP-1 number 3 is avoided because it have a single amino acid change.

3.4.2.2 Subclone PP4-1_B into pCAT-EYFP-2 vector

We would like to subclone PP4-1 cDNA to pCAT-EYFP-2 cloning vector in order to produce fusion-protein that will carry the EYFP tag on the C–Terminus. PP4-1_B that already amplified was digested using NotI and SacI restriction enzyme. The vector, pCAT-EYFP-2, was digested using NotI and SacI restriction enzyme as well. Those enzymes will cut specific recognition sites in the vector in order to make it linear. Both PP4-1_B and pCAT-EYFP-2 have to be treated with same restriction enzymes that create compatible ends.

The PP4-1_B was ligated into cloning vector, linear pCAT-EYFP-2. Following the method of ligation, reagents for each ligation mixture was prepared. The ligation mixture was left at 4^oC overnight before it was used in transformation of bacterial cells (Escherichia coli JM109) and transferred to LB agar plate containing Ampicillin. Selection of bacteria with ampicillin resulted survival of cells that have successfully inserted by plasmid. Ten colonies from this plate were selected and PCR colony was performed to check those colonies (Figure 3-41).

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Figure 3-41. The result of gel electrophoresis of colony PCR PP4-1 into pCAT-EYFP-2 vector. The size of expected PCR product is about 1.0 kbp. Colony PCR of PCR pp4-1 with vector pCAT-EYFP-1 showed all positive colonies.

The Primers were AK74f as forward Primer and AK94r as reversed Primer.

According to the positive result from Figure 3-41, two colonies from PP4-1_B--pCAT-EYFP-2 plate (number 1 and number 3) were selected and cultivated in LB Broth overnight. Plasmid isolation was performed using the plasmid miniprep kit and the concentration was measured. This plasmid was sent for sequencing. Table 3-22 provides the detail of mixture plasmid and selection primers that were used. The sequencing result is shown in Appendix 3-5.

Table 3-22. The premixture of PCR PP4-1_B--pCAT-EYFP-2 plasmid for sequencing.

Sample Plasmid Plasmid (µL)

Water

(µL) Primer (3 µL) Sample Sequencing Name PP4-1_A--pCAT-EYFP-2

number 2

1.0 11.0 AK93f PP4-1DECR_2AK93f_AK93f 1.0 11.0 Ak94r PP4-1DECR_2AK94r_AK94r PP4-1_A--pCAT-EYFP-2

number 7

1.7 10.3 AK93f PP4-1DECR_7AK93f_AK93f 1.7 10.3 Ak94r PP4-1DECR_7AK94r_AK94r

According to the result in Appendix 3-5, all sequences clones of PP4-1_B--pCAT-EYFP-2 are correct and can be used for subcellular localization.

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3.5 Subcellular Localization Studies of PP4-1, PP4-2, PP4R2L, and