Adventurous Manufacturing Follow On: Integrating Living Analytics into Biomanufacturing Processes

细胞拥有各种机制来感知环境，并通过改变自己表达的蛋白质来适应变化。使用合成生物学方法，有可能将这些方法结合起来开发分析工具，以报告生物制造中研究人员感兴趣的关键代谢物的浓度。我们称这些分析工具为全细胞生物传感器。然而，开发全细胞生物传感器只是挑战的一部分。为了能够将它们用于生物制造，我们需要一种方法来限制全细胞生物传感器的生长，并防止它们在制造过程中与其他细胞发生负面作用。在第一轮的概念验证赠款中，我们展示了将全细胞生物传感器封装在由多层组成的聚合物球中，使它们能够保持其生物传感能力，并与哺乳动物细胞一起生长，生成蛋白质产品，而不会对哺乳动物细胞的行为产生负面影响。在这项研究中，我们的目标是扩大这些发现，并与我们的工业合作伙伴一起，展示这一概念在工业生物制造中的潜力。拟议研究的第一部分将对我们最初的概念验证进行调整，使其更符合工业用途。这将涉及对生物传感器电路进行一些更改，以使输出更容易解释，并看到在更快的时间范围内发生的变化。它还将涉及使我们的聚合物球变得更小，以便它们与用于测量荧光的仪器更兼容。最后，我们将展示该生物传感器可以用于生物反应器中，在那里它与表达蛋白质的哺乳动物细胞一起生长。拟议研究的第二部分将开发一个概念，其中生物传感器不仅报告分析物的浓度，而且超越这一点，通过酶的表达来调节浓度。这将把控制制造过程的两个步骤--传感和驱动--结合到生物传感器中。要做到这一点，我们将修改我们的原始设计，以便细胞表达能够在检测到分析物时代谢分析物的酶。我们还将测试不同类型的聚合物和不同形状(球体除外)是否在此应用程序中工作得更好。最后，我们将在生物反应器中尝试该系统，并测试生物传感器每单位面积能降解多少聚合物代谢物。这将使在更大范围内使用这一概念的可行性成为可能。拟议研究的第三部分将建立在迄今交换用作生物传感器宿主的生物体类型的工作基础上，并表明该概念可以适用于我们最初例子之外的其他分析物。在这里，我们将探索食品加工中使用的生物体，以及与人类宿主共生且对人类无害的自然细菌。将探索的其他分析物已经被选择来代表广泛的化学结构，以评估我们可以检测到的分子类型是否有任何限制。到项目结束时，我们将进一步发展我们的初始概念，并促进其转化为工业。我们已经成功地展示了一种分析技术的新概念，我们认为这一概念可以对生物制造过程的分析和控制产生持久的影响。

Cells possess a variety of mechanisms to sense their environment and adapt to changes by changing the proteins they express. Using synthetic biology methods, it is possible to co-opt these methods to develop analytical tools to report the concentrations of key metabolites of interest for researchers in biomanufacturing. We call these analytical tools whole-cell biosensors. However, developing the whole-cell biosensor is only one part of the challenge. In order to be able to use these in biomanufacturing, we need a way to limit the growth of the whole-cell biosensor and prevent them from having negative interactions with the other cells in the manufacturing process. In a proof-of-concept grant from Round 1, we showed that encapsulation of the whole-cell biosensor in polymeric spheres made of multiple layers allowed them to retain their biosensing capability and be grown together with mammalian cells making a protein product without negatively affecting the behaviour of the mammalian cells. In this research, we aim to expand on these findings and, together with our industrial partners, demonstrate the potential of this concept in industrial biomanufacturing.Part 1 of the proposed research will make adjustments to our initial proof-of-concept to make it more compatible with industrial use. This will involve making some changes to the biosensor circuit to make the output easier to interpret and to see changes that occur in a faster time frame. It will also involve making our polymer spheres smaller so that they are more compatible with instruments used to measure fluorescence. Finally, we will show that the biosensor can be used in a bioreactor where it is grown alongside mammalian cells expressing a protein.Part 2 of the proposed research will develop a concept where the biosensor does not just report the concentration of the analyte, but goes beyond this to adjust the concentration via the expression of enzymes. This will combine two steps in the control of manufacturing processes-sensing and actuation-into the biosensor. To do this, we will modify our original design so that the cells express enzymes that can metabolise the analyte when it is detected. We will also test whether different types of polymer and different shapes (other than spheres) work better in this application. Finally, we will try the system in a bioreactor and test how much metabolite the biosensor can degrade per unit area of the polymer. This will inform the feasibility of using this at larger scales.Part 3 of the proposed research will build on the work so far to exchange the type of organism used as the host for the biosensor and show that the concept can work for other analytes beyond our initial example. Here we will explore organisms used in food processing as well as natural bacteria that live with and are not harmful for human hosts. The additional analytes that will be explored have been chosen to represent a wide range of chemical structures in order to assess if there are any restrictions on the kind of molecules we can detect.By the end of the project, we will have developed our initial concept further and facilitated its translation into industry. We have successfully demonstrated a new concept in analytical technology that we think can have a lasting impact on how biomanufacturing processes are analysed and controlled.