20.109(S07): Start-up expression engineering

Introduction

From your work so far this term, you have a good understanding of (at least) two fundamental concepts. From our first experimental module it should be clear that the genetic program for running a cell is readable (through sequencing), writable (through molecular biological techniques and synthesis) and somewhat, though not perfectly understandable. Recall how a genetic part can be as small as 13 base pairs, BBa_B0032 for example, to help carry out the work of protein production by enabling a huge protein/RNA complex, the ribosome, to bind an RNA molecule. From our second experimental module, it should be clear that a cell's programming doesn't end at protein production but rather that proteins are dynamic (chemically and spatially). They react to changes in the envirnoment with great speed and sensitivity. We saw that proteins may be digital information, either present or absent, but as far as the cell is concerned, they are tunable activities to be regulated by stability, localization and modification. Thus, from the work we've done so far this term, you may have the idea that gene expression in a cell is driven by the central dogma (DNA making RNA making protein) and regulated by what goes on with the protein after it's made. However, nature has refined this model for genetic programming still further, so a cell's proteins are not all constitutive (i.e. produced all the time) but rather their expression is regulated. With this module we will consider gene regulation at the level of transcription in a eukaryotic cell. SAGA intro 1. Gene expression in euk intimately related to chromatin dynamics 2. SAGA is 19 subunit complex req'd for chrom remodel and appropriate gene expression 3. Only 6 of SAGAs 19 subunits essential for viability. 4. Your mission, should you choose to accept it....

Protocols

Part 3: PCR

1. pRS406 template

DONE!