The Development of a New Cloning Strategy and the Demonstration of its Feasibility

5.1 Overview During the course of our experiments constructing the PromGlun diblock in Chapter 3, it became evident that a different approach was necessary to attain our goal of producing high molecular weight surface-active PAA’s. The limiting factor was that the conventional cloning techniques used did not permit us to construct the long DNA sequences that were required to encode for a higher molecular weight tail block. Thus, we could not produce a PAA diblock with a sufficiently long tail block for optimum brush extension. Therefore, alternative cloning strategies were investigated. In the literature, several techniques have recently been developed for the production of synthetic structural protein polymers. We considered each technique to determine if it could be applied to the production of our PAA design. Although these techniques were successful for their specific applications, we found they were still inadequate for our purposes. Most of these techniques relied on a limited pool of restriction enzymes, inefficient reactions (e.g. head-to-tail self-ligation of short DNA blocks), and required numerous experimental steps. These methods were also relatively complicated and provided a low level of control and flexibility. The attempt to apply one of these methods towards the construction of a gene encoding for a high molecular weight PAA diblock is presented in Section 5.3. After several failed attempts to apply these techniques, we concluded that we must develop a new cloning strategy. Section 5.4 presents the development of a new universal cloning technique for the streamlined production of surface-active PAA’s that enables a high level of control over polymer composition and molecular weight, while also being relatively simple and flexible. Chapter 6 will present how we applied this method to the production of a unique surface-active PAA designed to form brush layers on aluminum oxide surfaces. A manuscript submitted to Biomacromolecules based partly on the work presented in this chapter is located in Appendix A.