Project's background part II

     The power plants and railways were removed. During the washing, the staff intentionally left part of this tower intact, and made an contrast for further research of anti-contamination and anti-weathering (Figure 5-3, 5-4)

    However, 2015 field research shows most external lithic murals, statue, and towers still suffer from damage. Some even have distraction in main stone body. Meanwhile, there grows moss. Indoor stone inscriptions though are visible; there are obvious and irremovable cracks.

     Today, most preservation of lithic relics employ physical, chemical, or engineer approaches no matter where. It has been proven by reality that each of those approaches has fatal disadvantages for long-standing preservation of lithic relics, and those disadvantages would even lead to the ruin of lithic relics.

    Since forever, there are two categories of methods to preserve-- organic and inorganic. Organic materials cannot reach the standard of time to preserve relics such as epoxy resin, acrylic resin, silicone, etc.; inorganic materials would form hard shell that is not compatible or even react with the lithic relics, such as lime, barium hydroxide, and sulphuric acid.

     In conclusion, finding a long-standing, inexpensive, and efficient method to preserve lithic relics is an impending issue.

Figure 5-3

Figure 5-4

Principles of experiments

    The documents cited shows that even after natural weathering, biological decomposition, and acid rain erosion for a time period of time, some stone inscriptions, which are on rock surface containing calcium, remain in good condition because there is a thin layer of naturally formed semitransparent mineralised film that is hydrophilic and compact on such rock surface.

     In some part under the fine film, knife scratches on the stone from over a thousand years ago are still visible indistinctly. According to solid analysis, natural mineralised film is primarily composed of calcium oxalate monohydrate [Ca(COO)2·H2O] whose process of composition is interfered by microorganism.

     There are some advantages of calcium oxalate being a film as follows,

        1) In water, especially in acidulous water, the solubility of calcium oxalate is one order of magnitude smaller than normal calcium carbonate.

        2) Organically oriented calcium oxalate crystal's particles are fine and compact, which prevent the infiltration of toxic and harmful materials.

        3) The system can't be easily broken by external forces in that there is no distinct boundary between calcium carbonate rock and calcium oxalate film.

        So, this research project has chosen E. coli that has been transcribed with lipase to clean some stone relics that are covered with oil; then, we use E. coli, with transcription, that can secrete oxalate acid upon the stone relics. Therefore, there forms calcium oxalate to be protection film in order to preserve lithic relics.

        If we use chemical methods to generate calcium oxalate, we have to immerse the lithic relics in the chemical liquid; however, it is not suitable for the preservation of large-scale outdoor lithic relics. In fact, the natural existing calcium oxalate comes from the reaction of oxalate acid generated by microorganism and calcium compound on the surface of stone. This research project will utilise genetic engineering methods to "graft" the gene that generates calcium oxalate into Escherichia coli (E. coil).

    Then oxalate acid reacts with calcium lithic relics to generate calcium oxalate, which stimulates its natural formation process. Finally, there forms a fine layer to protect the lithic relics.

Procedures in details

     4 实验过程

    4.1 实验试剂及仪器

    4.1.1 目的基因 草酰乙酸乙酰基水解酶（oxaloacetate acetyl hydrolase 简写OAH）是本实验所使用的目的基因片段，主要功效是在表达时将草酰乙酸转化为草酸，草酸进而于钙结合生成草酸钙起到文物保护的作用。此目的基因片段从基因库中查的基因序列后由美国IDT公司直接合成得到。

    4.1.2 菌种和质粒 本实验使用的质粒为pET-28a，使用的大肠杆菌为TOP-10感受态细胞，购于天根生化科技有限公司

    4.1.3 酶与试剂 实验所使用的限制性内切酶购于NEB，连接酶购于天根生化，DNA纯化试剂盒以及质粒提取试剂盒均购于天根生化。

    4.1.4 PCR引物 根据OAH以测得的序列，由Invitrogen合成了如下引物：

    上游引物：5’-ATCATCTTGCATATGGCGTCCACCATTGCTGTT-3’（划线部分为Nde i酶切位点）

    下游引物：5’-TTTCCTTTTGCGGCCGCTTAG-3’（划线部分为Not i酶切位点）

    4.1.5 培养基 实验使用的培养基为LB，以配备200ml的量为例，需要如下材料：胰蛋白胨2g，酵母提取物1g，NaCl 2g，加水至200ml。

    实验使用的培养板加入卡那抗性，一配备400ml为例，需要以下材料：胰蛋白胨 4g，酵母提取物 2g，NaCl 4g，Agar琼脂粉6g，卡那霉素 1.2ml

    4.2 实验步骤

    4.2.1 PCR扩增 因为目的基因片段的G-C碱基对含量很高，所以PCR扩增时需使用GC Buffer，参照Takara产品目录配200ul体系：GC buffer 100ul,dntp mixture 16ul, 前引物 4ul, 后引物 4ul,DNA模板 4ul, Taq 2ul, H2O 70ul。用60℃-70℃进行梯度扩增，寻找引物合适的退火温度。发现在第六条带最亮，再按照上述配成新的体系在适宜温度下进行PCR扩增，得到加上接头的基因片段

    4.2.2 做完PCR之后，因有较多的杂条带，故将所有扩增得到的产物跑胶，将所需片段切胶纯化。

    4.2.3 进行酶切，分别在目的基因片段和载体pET-28a中加入Not I 和 Nde I 的限制性内切酶。

    4.2.4 进行连接并设置对照组。实验组：大片段＋小片段＋T4 DNA Ligase＋Buffer

    对照组：大片段 在16℃的环境下连接过夜。

    4.2.5 使用DNA纯化试剂盒提取连接后产物，转化进TOP-10感受态细胞，涂在配置好的带卡那抗性的培养板上，放在37℃的恒温环境下培养。

    4.2.6 根据菌落的生长情况，将实验组的细菌进行质粒提取，并再次用上述的两种酶进行酶切后跑胶，如果跑胶后大片段和小片段的位置正确，则连接完成。

    4.2.7 测序。

    4.3 实验结果