Introduction Materials and Methods .1 Place and time of research

III. CLONING TREHALOSE SYNTHASE GENE from Thermobifida fusca and Mycobacterium tuberculosis

3.1 Introduction

This chapter concentrated to provide the clone of the gene TreS within cloning vector and within plant expression cassette as a preparation to express the gene in Arabidopsis thaliana to assess the effect of the gene in plant responses to stresses. There are many TreS bearing organisms, such as bacteria and fungi. The gene that is going to be cloned, would be expressed in the plant, hence, enzymes that active in the condition about similar to the cellular plant condition would be favorable. Few enzymes with their condition for optimum activities are presented in Table 1. Codon usage is also necessary to be considered to maximize the expression of the gene. Some comparisons of codon usage are presented on appendix 1. Information was gathered regarding to the characterisstics pH, temperature, Km of trehalose synthase from few organism that has been reported. It is shown on appendix 1 that among trehalose synthases reported, those with low Km and high maximum conversion rate at the condition suitable in the plant cells would be favourable to be express in plant. TreS gene from Thermobifida fusca , has optimum temperature of 25 and pH 6.5 and it’s activity is not affected by the concentration of substrate Wei et al. 2004. Hence, this enzyme was chosen to be expressed in Arabidopsis thaliana. It was also cloned TreS from Mycobacterium tubercolusis Murphy et al. 2005 as it’s sequence is very similar to that of Thermobifida fusca. 3.2 Materials and Methods 3.2.1 Place and time of research This experiment was conducted in The Department of Molecular Plant Physiology, Utrecht University, Netherlands, started from September 2004-March 2005.

3.2.2 Materials

TreS gene source, lyophilized Thermobifida fusca cat No. 43792 was purchased from Deutsche Summlung von Mikroorganismen und Zelkulturen DSMZ, Germany and TreS gene from Mycobacterium tuberculosis inserted in plasmid pCR Blunt II TOPO was kindly donated from B. Rebertson Imperial Collage, London, UK.

3.2.3 Cloning of TreS genes from Thermobifida fusca and from Mycobacterium tuberculosis

Growth condition of Thermobifida fusca and DNA isolation Lyophilized cells of the bacteria received in tablet was dissolved in sterile water and spread on top of solidified 65 medium pH 7.2 [glucose 4 gL; yeast extract 4 gL; malt extract 10 gL; CaCO3 2gL omit if liquid medium is needed and 12 gL agar]. The culture was incubated at 55 o C as suggested by DSMZ. When grown cells were obtained, DNA was isolated using plant DNA extraction kit, Pure Nucleophyton Qiagen. Cloning strategy TreS gene was isolated using PCR cloning approach with forward primer: 5’-GAG CCA TGG AGA AGT CGA TGA CCA C-3’ and reverse primer: 5’-TGG AAG CTT TCA GGA CG CTG GGT C-3’. These primers were spanned outside the TreS gene within the genome. Optimum annealing temperature was determined using PCR program with gradient annealing temperature as follow: 1x cycle of 94 o C for 5 , followed by 40x cycles of 94 o

C, 30’’; 45-60

o C, 30’’ and 72 o C, 2’; then 1x cycle of 72 o C for 5’. The PCR product reaction mixtures then were run on 0.8 gel electrophoresis at 90 volt for about 30 minute. The gel was subjected to UV transiluminator to see the DNA fragments on the gel. The optimal temperature determined from the gel was used for all PCR work. The right fragment of PCR product was eluted using DNA gene clean kit QIAEX II Qiagen. The eluted DNA then was inserted into pGemT Easy Vector System I Promega with ligation mixture: 2X ligation buffer 4 µ L, eluted DNA 2 µ L, pGemT vector 1 µ L and enzyme T4 DNA lygase 1 µ L. After over night incubation at room temperature, the mixture then was transformed into competent cells of E. coli DH5 α using Heat Shock transformation method. Ligation mixture of a 1.5 µ L was added to micro tube contained of 100 µ L competent cells CaCl 2 and incubated in ice for 1 h before transferring to 42 o C water bath for 2 minute. The tube then was immediately transferred to ice and kept for 15 minutes. Luria Broth LB medium was added to the tube, incubated at 37 o C for 1 h. A 100 µ L of suspension cells was plated on solidified LB medium supplemented with 100 mgL amphycilin. The rest of suspension cells were centrifuged and about 100 µ L of supernatant was kept for resuspension before plating on the same medium. The culture was incubated at 37 o C, and colony number was recorded after 24 h. To verify whether the colonies bring the right plasmid with the right gene, 6 colonies were reanalyzed. These colonies were grown on 3 ml liquid culture supplemented with 100 mgL amphycilin and incubated at 37 o C for overnight. Plasmid was then re-isolated using alkali lysis Sambrook and Russel, 2001 and digested using EcoRI to see the right size of the gene, or using PstI to see the orientation of the gene and also to give another prove for the right gene. Two clones among those that contained the right TreS gene were re-grown in the same medium and the plasmid were re-isolated using Jet Quick Miniprep Spit Kit Qiagen to provide a high quality of plasmid DNA. DNA concentration was determined using spectrophotometer at 260 nm. The DNA then was sequenced with the sequencing mixture as follow: DNA plasmid 1.2 µg, 10 pmol Primer forward as used to obtain the gene or 10 pmol M13 primer and then adjusted volume using deionized water to 16 µ L final volume. To obtain the whole gene sequence forward or reverse internal primers were also used. This mixture then was sent to commercial sequencing company at Utrecht University. Verifying TreS from Mycobacterium TreS gene from Mycobacterium that has been inserted in plasmid pCR Blunt II TOPO was kidly donated from B. Robertson. The plasmid was retransformed into E.coli, and after overnight culture the plasmid was re-isolated from cells. Restriction analysis was performed to recheck whether it bears the right gene. Digestion using NdeIXhoI was done to check the size of the gene and Bam HI or EcoRI to see the orientation of the gene and give more prove for the right gene. The digestion mixture was then run on 0.8 gel electrophoresis at 90 volt for about 30 minutes. Insertion of TreS gene to Plant Expression Vector Plant expression vector used was pBIN1935S provided by Utrecht University. This plasmid was derived from pBin19 that all the multiple cloning site has been removed using HindIII and EcoRV. The sticky ends resulted then were filled using klenow fragment to produce blunt ended fragment. Promoter of 35S and multiple cloning sites from pUC19 was excised from PUC19 using EcoRV, and klenow fragment was subjected to this fragment to obtain blunt ended fragment. pBin19 fragment and 35S promoter-multiple cloning site from PUC19 was then ligated to create pBIN1935S plasmid. TfTreS that was available in pGemT easy vector system I was digested using EcoRI and was eluted from gel using QIAEX II Qiagen. At the same time, pBIN1935S was digested using EcoRI and then was treated with alkaline phosphatase for 30 minutes. The gene fragment and the vector was ligated for overnight, followed by transformation to CaCl 2 E.coli competent cells using heat shock transformation method as the same method mentioned before. The cell suspension then was plated on agar solidified LB supplemented with 50 mgL kanamycin. Colonies obtained was observed after over night culture and 20 colonies were chosen to be re-analyzed whether they have the right plasmid with the right gene at the right orientation. Plasmid isolated from over night liquid culture then was digested using EcoRI to see the size of the gene, and using Bam HI to see the orientation of every clone. Plasmids that produced about 2000 bp fragment when digested with EcoRI and about 1100 bp fragment when digested using BamHI were the expected one. MtTreS was exciced from plasmid PCR Product Blunt II TOPO using Xba ISacI. The digestion mixture was verified on 0.8 gel electrophoresis. Fragment DNA about 2000 bp was eluted from gel, and ligated with pBIN1935S that has been digested with XbaISacI and treated with alkaline phasphatase for 30 minutes. The ligation mixture was transformed into E.coli, and the clones obtained were reanalyzed using BamHI.

3.3 Result and Discussion