Molecular Techniques in Biosciences (D224P6 MY)

______________________________________________________________________________
26 MARCH 2013

Evaluation of tissue culture and particle bombardment procedure for Sauropus androgynus and plasmid DNA transformation from Escherichia coli through alkaline lysis.
By student ID 009921
School of Biosciences, University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor.

Abstract Different plant species might require different protocol to be used when culturing and performing particle bombardment. The aim of this study is to test the protocols for tissue culture, plastid DNA extraction and isolation from Escherichia coli and biolistic parameter for Sauropus androgynous. Surface sterilisation used in this study were inadequate to prevent fungual and bacterial contamination but the contamination still low at 16.67%. Alkaline lysis method used to obtained plasmid DNA from Escherichia coli are heavily contaminated with 2.19
ratio of A260/A280 from nanodrop and not able to be used for biolistic. Particle bombardment

procedure was done through Biolistic PSD-1000/He and successfully produced organogenesis (roots) and healthy growing callus.

009921

1.0 INTRODUCTION The recombinant technology has allowed the researchers to break the gene barrier to created transformed or genetically modified (GM) organism to fulfil some beneficial purpose like enhanced nutritional value, pest resistance, increase yield and many other examples. Many protocols have been established in order to find a better way to deliver the recombinant DNA like particle bombardment. Particle bombardment or biolistic, utilize high velocity microprojectiles to deliver foreign DNA into the target cells or tissues. The original concept come from Sanford et al. (1987) whom invent the term itself biolistic, which stand for biological ballistics. It is a versatile methodology to deliver foreign DNA particularly plasmid because of its wide range of target cells from diverse organisms. However different organism might have different parameter to be used for particle bombardment and this paper is taken an approach to test the parameter for the targeted plant. Plasmid transformation has been first established on as early as 1980s by Boynton et al. (1988) in Chlamydomonas reindhartii. Since then, many researcher has applied the technology and became popular approach due to their own transcription-translation machinery. Plasmid is usually obtained from bacteria and plasmid DNA extraction and isolation is not an easy task to be carry out. Hence, many protocols have been developed to enhance the quality of extracted DNA plasmid. In this writing, an approach is taken using alkaline lysis to observe the successfulness of the method to extract and isolate plasmid DNA from Escherichia coli. The chosen target plant for the transformation is Sauropus androgynus because of its nutritional values. S. androgynous is an underutilized green herb and known among the local Malaysians as cekur manis or asin-asin. Many researches of this plant is related to Bronchiolitis Obliterans (BO) syndrome that cause an outbreak at Southern Taiwan on 1995 (Ger et al. 1997). Unfortunately, not many researches have been done related to nutrition aspect of the species. One study (Subhasree et al. 2009) proved that the plant is a rich source of antioxidants like vitamin A, B and C. This paper is aimed to prepare the culture of S. androgynous through indirect somatic embryogenesis. Here surface sterilization is also tested to see either the plant survive or not than can be observed by the growth of the culture. The developed culture then further undergoes plastid transformation using plastid obtained from E. coli. Plasmid extraction and isolation procedure is done through alkaline lysis. For this purpose, the recombinant protein

009921

candidate is green fluorescent protein (GFP) and done through biolistic transformation where the parameter will be tested parameter to suit the culture.

2.0 METHODOLOGY 2.1 Establishment of aseptic culture of S. androgynus Healthy juvenile leaves of S. androgynus is selected from the shade house and washed using tap water to remove dirt or any surface contaminants. The washed leaves are cut into 12 pieces with size approximately 1.5 cm x 1.5 cm using scissors. The excised leaves then undergoes surface sterilization, the sterilants used are; 70% (v/v) ethanol solution and 20% (v/v) sodium hypochloride all performed in sterile Schott bottle for 20 minutes to reduce risk of contamination. The damaged area or edges that cause by the sterilants is trimmed into not larger than 1 cm x 1 cm. The six trimmed explants then transferred into each petri dish with MS + 2 mg/l NAA and 1.0 mg/l Kinetin with the adaxial surface of each explants is gently pressed against the agar to ensure a good contact. Each petri dishes are sealed with parafilm to maintain moisture and incubated at 25oC in 16 hour light / 8 hour dark for 14 days. After 7 days, the explants are examined for contamination. If there is a contamination, subculturing is needed for non-contaminated explants by transferring into other sterile petri dish with the same medium. After 14 days, the culture will undergoes biolistic. 2.2 Isolation and purification of plasmid DNA For plasmid extraction, alkaline method is used based on protocol developed by Birnboim et al. 1979. A single, well-isolated colony of Escherichia coli from a recent sub-cultured Luria Bertani (LB) agar plate is inoculated and incubate overnight (not longer than 16h to prevent plasmid loss) at 37oC. 10ml of the bacterial cultured is centrifuged and the pellet is resuspended in pre-chilled Solution I and incubate at room temperature. A fresh prepared Solution II is added and gently inverted before incubated on ice. Then, a pre-chilled Solution III is added and gently inverted several times before incubated on ice. The solution is centrifuged. The top phase is transferred to the new tube and phenol-chloroform-isoamyl alcohol (25:24:1) is added before centrifuge again. The top phase is transferred into a new tube and incubate at room

009921

temperature after 100% absolute ethanol is added. The solution is centrifuged and the pellet is washed with 70% pre-chilled ethanol incubate at least 15 minutes at -20OC. After the incubation, the solution is centrifuge, supernatant is removed and let the pellet dry. The pellet then resuspend in TE buffer. Spectrophotometer is used to quantify the amount of DNA obtained and gel electrophoresis is done to identify any contamination. 2.3 Microprojectile-mediated DNA delivery into culture of S. androgynus Healthy explants that have most callus growing after 2 weeks of culturing is chosen for biolistic. The device used is Biolistic PSD-1000/He and sterilized by wiping the apparatus and biolistic chamber with 70% (v/v) alcohol. Gold microcarrier 0.6 in size is also being sterilized by washing in 100% ethanol suspension and vortex. The supernatant is removed and washing step is repeated for two before resuspend on sterile water. 5 g/l DNA is added into the gold suspension and vortex at maximum speed. Calcium chloride and spermidine is added. The mixture is centrifuge and the supernatant is discarded. The pellet is resuspended in 100% (v/v) ethanol and vortex at maximum speed for 1 min and centrifuge again. The supernatant is discarded before resuspended in 100% (v/v) ethanol. Particle bombardment done at 9 cm target distance at 1100 psi with vacuum pressure used 26 in.Hg. The culture is left for a week and observed for any changes in growth and contamination.

3.0 RESULTS AND FINDINGS 3.1 Establishment of aseptic culture of S. androgynus After a week, not all of the explant shows distinctive callus growth. Callus appear to be clear in colour with irregular shape appeared at the edge of the explants. There is 16.67% of contamination of fungus on the explant and one contamination on medium for both petri dishes (refer Figure 1.0). Browning colouration occurred as high as 75.0% (nine explants) for both petri dishes. On average, 56.3% of the surface area of the explants shows browning. Subculture is done to save the five selected explant that sterile or free from contamination to the new plate and no contamination of any kind observed after a week. The culture then is chosen as candidate for biolistic.

009921

D
G1
G1 C
1

C
2

G2 G2

C2

Figure 1.0 : (A) Explants of S. androgynus in both petri dishes in day 1 (B) The contamination occurred in both petri dishes after a week. The arrow C1 indicateds the area of contamination of fungus on the growth media and arrow C2 indicates fungus contamination on the explant. There were some callus growth observed in some explants (G 1 and G2). (C) Week 2 of the subculture chosen from week 1 (that contain contamination of fungus). Explants in week 2 exhibit zero contamination both on media and explants (D) The close up of callus growth (G1 and G2) exhibit in some of the explant in week 1.

3.2 Isolation and purification of plasmid DNA Plasmid extraction and isolation is done using alkaline lysis method. The results of gel electrophoresis shows neither smearing nor present of any DNA (refer Figure 1.2, well M4). However the ratio of A260 to A280 (260/280) from nanodrop result shows 2.19 (refer Table 1.0) indicates there is contamination of proteins probably from RNA (ratio 1.8 indicates pure DNA). In this experiment, the methodology give high amount of contamination and unable to give the desired DNA amount. The DNA obtained did not used for the biolistic due to these limitation instead using the prepared DNA sample from the lab.

009921

Table 1.0 : Nandrop reading obtained from spectrophotometer. Absorbance at 260nm (A260) shows the DNA concentration and absorbance at 280nm (A280) indicates the protein contamination. A good quality DNA should have a ratio 260/280 about 1.7-2.0, larger value indicates the contamination of proteins and other molecules.

Sample ID 4

ng/l 203.51

A260 4.070

A280 1.858

260/280 2.19

Figure 1.3 : Gel electrophoresis of the plasmid DNA (well M4) shows unclear smearing and no clear DNA fragments is being detected. Lambda Hind III was used DNA ladder.

3.3 Microprojectile-mediated DNA delivery into culture of S. androgynus Post biolistic explant shows more browing in colour compare to prebiolictic (refer Figure 1.1). Observation showed most of them exhibit somatic embryogenesis callus formation and one explant exhibit organogenis of roots. Calluses at this stage are more distinctive with whitish colour in irreglobular structure aroung the edges of the explants. No contamination was observed.

009921

Figure 1.1 : (A) Explants of S. androgynus arrangement before biolistic (B) Explants a week after particle bombardment shows browning in colour. Organogenesis of roots (arrow R) and more callus growth (arrow G) can be observed in every explant compare to pre-biolistic. (C) Close up picture showing the organogenesis of roots.

4.0 DISCUSSION 4.1 Establishment of aseptic culture of S. androgynus Contamination that occurred after a week in tissue culture is also probably due to inadequate aseptic technique and lack of surface sterilisation. Inadequate aseptic technique usually happen when the tissue culturist is lack of skills and experiences to sterile the equipment properly and lack of proper equipment (in example; face mask). Contamination is transmitted mainly through airborne and contact. It is encourage for the handler to regularly clean and sterile the equipment and work place especially before and during the process of culturing with 70% ethanol and reduce verbal communication during culturing or wear face mask. With the adequate aseptic technique, the improvement can be shown with subculture result where no contamination of fungus or bacteria was observed. Surface sterilisation used in this experiments was; 70% (v/v) ethanol solution and 20% (v/v) sodium hypochloride for 20 minutes. The culture observed during the period of the experiment grow very well but contamination occurred only in first week of the culture growth. It is assume that the surface sterilants used is compatible for the culture to grow but inadequate

009921

for removing the contaminants. It is suggested to sterilize the plant for longer period of time or used mercuric chloride (HgCl2). One study (Ramakrishna et al., 1991) proved the usage of HgCl2 can significantly reduce the risk of many bacterial and yeast contamination including Alternaria alternata, Fusarium spp. Aureobasidium pullulans, Bacillus spp., and Epicoccum purpurascens, when used for 3-10 minutes. 80% (v/v) of ethanol concentration can be used instead 70% (v) in this experiment as one study able to get as high as 90% aseptic culture (Giladi et al., 1979). However, both suggested method might reduce the culture growth as shown in both studies. In this experiment however, the contaminants can only be suspected because no approach was taken to identify the contamination and contamination within the tissues remains unknown. It is suggested to take a safety precaution to culture the explants with antibiotics and antifungal to prevent bacterial and fungal growth. The antibiotics rifampicin, tetracycline, cefotaxime and polymyxin B are the four proven antibiotics known to be effective against bacteria in tissue culture (Young et al., 1983).

4.2 Isolation and purification of plasmid DNA Alkaline lysis for plasmid extraction required certain crucial solution which represent by Solution I, II and III. Solution I contains glucose, Tris-HCL (pH 8.0), EDTA (pH 8.0) and autoclave at 121C for 15 min stored at 4C. EDTA or ethylenediaminetetraacetic acid plays an important role in Solution I to stabilize DNA phosphate backbone and chelate divalent metal cation (Mg2+ and Ca2+) for DNAses thus prevent it from function properly. In other words, EDTA protects the DNA by deactivating metallopeptidase or metal-dependant enzyme including DNAses from digesting the DNA by chelating the metal ion that required by the enzymes (Auld, 1995). Another effective method is pretreatment of the explant into antibiotic achromycin that could control up to 100% contamination at 100 mg/l (Geier, 1997). Solution II is also alkaline as it contains Sodium hydroxide (NaOH) and 1% (w/v) Sodium dodecyl sulphate (SDS). SDS have hydrophobic properties that able to breakdown the plasma and nuclear membrane and destroy the histones conformation by removing the negative ions from the proteins. Meanwhile NaOH will denature plasmid and chromosome into single strands. Solution III is an acidic solution to neutralize the previous solutions used. It contains potassium

009921

acetate, glacial acetic acid and water. The main purpose of the Solution III is to aggregate the unwanted molecules like bacterial DNA and RNA, proteins, contaminants, and others so that it can easily discarded. The action of potassium acetate and acetic acid help to precipitate the dodecyl sulphate (from SDS) bound proteins. Manual extraction protocol have been used required skilful and experienced handler to be performed. Findings from nanodrop reading shows that the DNA obtained may heavily contaminated with proteins and RNA. One limitation using the nanodrop is the absorbance at 260nm indicates both concentration of both plasmid and genomic DNA and also RNA so the reading will not able to give the true concentration of DNA. However, gel electrophoresis have been done to justify the contamination of RNA, proteins and genomic DNA. One quirk is, the result of gel electrophoresis does not shows any smearing that supposed to indicate the present of the contaminants. It is suspected poor pipetting technique is man cause of this situation especially when one is doing manual extraction (will discuss below). It is suggest for DNA extraction and isolation to use kit that may give better result compare to manual. Another improvement that can be done is to replace NaOH with arginine buffer (in range pH 11.4 to 12.0) that proved to aid to obtain plasmid DNA suitably pure for both sequencing and also stabilize pH during the lysis (Cloninger et al., 2008). Handling technique is probably the most crucial part when conducting experiment. Pipetting technique applied in this experiment is in doubt since the results of gel electrophoresis and 260:280 from nanodrop indicates contamination of other proteins or RNA. Pipette is a precision instrument that a small mistake (especially in liquid handling) can lead to the large error in final results. The pipette tip should be prewet first with the aliquot of the sample solution at least three time. One study (Zeman et al., 1974) proved that without prewetting, an error up to 15% of rated volume dispensed from pipette. When aspirating the solution, the plunger should depressed until the first stop and when dispensing the solution, the plunger need to slowly depressed past the first stop to ensure the whole delivery of the solution.

009921

4.3 Microprojectile-mediated DNA delivery into culture of S. androgynus In biolistic, gold is preferable compare to tungsten because gold is homogenous, non-toxic and inert towards catalytic degradation (Sanford et al., 1993). Meanwhile tungsten might cause acidification of the agar medium that may lead to cell death thus destroying the culture (Sanford et al., 1993). 0.6 gold is chosen compare to 1.0 gold because small size microprojectile is more efficient for particle bombardment to give less tissue damage thus give a better chance of survival for tissue to grow (Randolph-Anderson et al., 1995). Theoretically, spherical shape particles should cause less damage to the cells. In this case, spherical structure of gold microprojectile may lessen the cell injury. Calcium chloride and spermidine used to help the adhesion of the DNA towards gold particles to be bombarded. Compare to tungsten, that have irregular structure can cause agglomeration during the DNA coating procedure while gold remain separated (Hunold et al., 1994). Based on the post-biolistic culture result, there were high amount of growth of calluses exhibit on the explants. This is a crucial findings because in study conducted using Nicotiana tabacum, gas blast and acoustic shock from the device introduced mechanical cell injuries that that take longer time to recover and even worst cannot be recover at all (Russell et al., 1992). The parameter used in this methodology proved to suit the S. androgynous culture. To further enhance the parameter, vacuum level can be increased to reduce more air friction that interfere with the bombardment. One study (Genga et al., 1991) shows the three fold increase using 28 in Hg of the beta-glucuronidase (GUS) reporter gene in tobacco. Browning that occurred during the growth of the culture and after particle bombardment is a neutral process that occurred due to oxidation of the phenolic compound that leached out from explants. Quinone that produced from oxidized phenolic compound are responsible for the discolouration (Murata et al., 2002). However, the products from phenols oxidation can be phytotoxic that lead to necrosis of the cells (Bhojwani and Razdan, 1996). Browning that occurred in the culture in this experiment is considered tolerable that may not cause severe necrosis of the tissue. In case the browning colouration needs to avoid or longer period time is needed for culture to growth, adding activated charcoal is proven to reduce this effect (Zhu et al., 1997).

009921

5.0 CONCLUSION This writing have successfully optimized some protocols while some needs further adjustment. Tissue culture of S. androgynus have been able to develop successfully and improvements on culturing mainly focus on how to reduce and identify the contamination. Future study on the effect of surface sterilisation towards the culture need to be done with proper aseptic technique applied so then it will not interfere with the results. Plasmid extraction and isolation through alkaline lysis was challenging especially to newcomer. Rigorous training and exposure on the essential laboratory technique seems necessary especially pipetting skill for one that will use this protocol. Contamination and unsuccessful attempt will cause time delay, increase cost and cause poor reputation of performer. Biolistic parameter of S. androgynus culture is probably the most crucial in this finding that can be further optimize to enhance the delivery of DNA. Further study could be done to further show the GFP gene expression in the culture. Another possible study that can be conducted is to optimize another parameters of the unsuccessful protocols so that any differences or similarities can be observed and finds which give a better results.

009921

REFERENCES

Auld, D.S. (1995) Removal and replacement of metal ions in metallopeptidases. Methods in Enzymology 248, 228-242. Subhasree, B., Baskar, R., Laxmi, R., Keerthana, R., Susan, L., Rajasekaran P. (2009) Evaluation of antioxidant potential in selected green leafy vegetables. Food Chemistry 115 (4), 12131220. Bhojwani, S. S., Razdan, M.K., (1996) Plant Tissue Culture: Theory and Practice. Studies in Plant Science (5), ix. Birnboim, H.C., Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Reseach 7 (6), 1513-1523. Cloninger, C., Felton, M., Paul, B., Hirakawa, Y., Metzenberg, S. (2008) Control of pH during plasmid preparation by alkaline lysis of Escherichia coli. Analytical Biochemistry 378 (2), 224-225. Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M. (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240, 15341538. Geier, T. (1977) Morphogenesis and plant regeneration from cultured organ fragments of Cyclamen persicum. Acta Horticulturae 78, 167174. Genga, A.A., Cerotti, A., Bollini, R., Bernacchia, G. and AIlavena, A. (1991) Transient gene expression in bean tissues by high-velocity microprojectile bombardment. Journal of Genetics and Breeding 45, 129-134. Ger, L.P., Chiang, A.A., Lai, R.S., Chen, S.M., Tseng, C.J. (1997) Association of Sauropus androgynus and Bronchiolitis Obliterans Syndrome: A Hospital-based Case-Control Study. American Journal of Epidemiology 145 (9), 842-849. Giladi, I., Altman, A., Goren, R. (1979) A method for aseptic culture of bud explants from citrus trees, Scientia Horticulturae 10 (4), 357-362.

009921

Hunold, R., Bronner, R., Hahne, G., (1994) Early events in microprojectile bombardment: cell viability and particle location. The Plant Journal 5, 593-604. Murata, M., Sugiura, M., Sonokawa, Y., Shimamura, T., Homma, S. (2002) Properties of chlorogenic acid quinone: relationship between browning and the formation of hydrogen peroxide from a quinone solution. Bioscience, Biotechnology, and Biochemistry 12, 2525-2530. Ramakrishna N, Lacey J, Smith JE. (1991) Effect of surface sterilization, fumigation and gamma irradiation on the microflora and germination of barley seeds. International Journal of Food Microbiology 13, 4754. Randolph-Anderson B, Boynton JE, Dawson J, Dunder E, Eskes R, Gillham NW, Johnson A, Perlman PS, Suttie J, Heiser WC (1995) Sub-micron gold particles are superior to larger particles for efficient biolistic transformation of organelles and some cell types. BioRad Technical Bulletin 2015, 1-4. Sanford, J.C., Smith, F.D., Russell, J.A. (1993) Optimizing the biolistic process for different biological applications. Methods in Enzymology 217, 483509. Zeman, G.H., Mathewson, N.S. (1974) Necessity of prerinsing disposable polypropylene pipet tips. Clinical Chemistry 20 (4), 497-8. Zhu, Y.-M. Hoshino, Y., Nakano, M., Takahashi, E., Mii, M. (1997) Highly efficient system of plant regeneration from protoplasts of grapevine (Vitis vinifera L.) through somatic embryogenesis by using embryogenic callus culture and activated charcoal. Plant Science 123 (12), 151157. Peter, M., Young, A., Anita, S., Hutchins, C., Marilyn, L. (1984) Canfield use of antibiotics to control bacteria in shoot cultures of woody plants. Plant Science Letters 34, 203-209.