Borg Yanika, Grigonyte Aurelija Marija, Boeing Philipp, Wolfenden Bethan, Smith Patrick, Beaufoy William, Rose Simon, Ratisai Tonderai, Zaikin Alexey, Nesbeth Darren N
Department of Biochemical Engineering, University College London, United Kingdom.
Department of Mathematics, University College London, London, United Kingdom.
PeerJ. 2016 Jul 7;4:e2031. doi: 10.7717/peerj.2031. eCollection 2016.
Aim. The nascent field of bio-geoengineering stands to benefit from synthetic biologists' efforts to standardise, and in so doing democratise, biomolecular research methods. Roseobacter clade bacteria comprise 15-20% of oceanic bacterio-plankton communities, making them a prime candidate for establishment of synthetic biology chassis for bio-geoengineering activities such as bioremediation of oceanic waste plastic. Developments such as the increasing affordability of DNA synthesis and laboratory automation continue to foster the establishment of a global 'do-it-yourself' research community alongside the more traditional arenas of academe and industry. As a collaborative group of citizen, student and professional scientists we sought to test the following hypotheses: (i) that an incubator capable of cultivating bacterial cells can be constructed entirely from non-laboratory items, (ii) that marine bacteria from the Roseobacter clade can be established as a genetically tractable synthetic biology chassis using plasmids conforming to the BioBrick(TM) standard and finally, (iii) that identifying and subcloning genes from a Roseobacter clade species can readily by achieved by citizen scientists using open source cloning and bioinformatic tools. Method. We cultivated three Roseobacter species, Roseobacter denitrificans, Oceanobulbus indolifexand Dinoroseobacter shibae. For each species we measured chloramphenicol sensitivity, viability over 11 weeks of glycerol-based cryopreservation and tested the effectiveness of a series of electroporation and heat shock protocols for transformation using a variety of plasmid types. We also attempted construction of an incubator-shaker device using only publicly available components. Finally, a subgroup comprising citizen scientists designed and attempted a procedure for isolating the cold resistance anf1 gene from Oceanobulbus indolifexcells and subcloning it into a BioBrick(TM) formatted plasmid. Results. All species were stable over 11 weeks of glycerol cryopreservation, sensitive to 17 µg/mL chloramphenicol and resistant to transformation using the conditions and plasmids tested. An incubator-shaker device, 'UCLHack-12' was assembled and used to cultivate sufficient quantity of Oceanobulbus indolifexcells to enable isolation of the anf1 gene and its subcloning into a plasmid to generate the BioBrick(TM) BBa_K729016. Conclusion.The process of 'de-skilling' biomolecular techniques, particularly for relatively under-investigated organisms, is still on-going. However, our successful cell growth and DNA manipulation experiments serve to indicate the types of capabilities that are now available to citizen scientists. Science democratised in this way can make a positive contribution to the debate around the use of bio-geoengineering to address oceanic pollution or climate change.
目的。新兴的生物地球工程领域有望受益于合成生物学家为使生物分子研究方法标准化并进而实现其普及化所做的努力。红杆菌属细菌占海洋细菌浮游生物群落的15 - 20%，这使其成为构建用于生物地球工程活动（如海洋废塑料生物修复）的合成生物学底盘的主要候选对象。诸如DNA合成成本不断降低和实验室自动化等发展趋势，在促进学术界和工业界等传统领域发展的同时，也继续推动着全球“自己动手做”研究群体的形成。作为一个由公民、学生和专业科学家组成的合作团队，我们试图检验以下假设：（i）能够完全用非实验室物品构建一个可培养细菌细胞的培养箱；（ii）使用符合BioBrick™标准的质粒，可将红杆菌属的海洋细菌确立为遗传上易于操作的合成生物学底盘；最后，（iii）公民科学家使用开源克隆和生物信息工具能够轻松地从红杆菌属物种中鉴定并亚克隆基因。方法。我们培养了三种红杆菌属细菌，即脱氮红杆菌、吲哚嗜盐菌和希氏玫瑰杆菌。对于每种细菌，我们测量了其对氯霉素的敏感性、在基于甘油的冷冻保存11周后的活力，并使用多种质粒类型测试了一系列电穿孔和热休克转化方案的有效性。我们还尝试仅使用公开可得的组件构建一个培养箱 - 振荡器装置。最后，一个由公民科学家组成的小组设计并尝试了从吲哚嗜盐菌细胞中分离抗寒anf1基因并将其亚克隆到BioBrick™格式质粒中的程序。结果。所有物种在基于甘油的冷冻保存11周内都保持稳定，对17μg/mL氯霉素敏感，并且在所测试的条件和质粒下对转化具有抗性。一个名为“UCLHack - 12”的培养箱 - 振荡器装置被组装起来，并用于培养足够数量的吲哚嗜盐菌细胞，以实现anf1基因的分离及其亚克隆到质粒中，从而产生BioBrick™ BBa_K729016。结论。生物分子技术的“去专业化”过程，特别是对于研究相对较少的生物体而言，仍在进行中。然而，我们成功的细胞生长和DNA操作实验表明了公民科学家目前所具备的能力类型。以这种方式实现普及化的科学能够对围绕利用生物地球工程解决海洋污染或气候变化的辩论做出积极贡献。
