The goal of synthetic biology is to make it easier to engineer biological systems. One important engineering tool for simplifying the process of engineering complex systems like biology is modularity. Standardized and modular biological parts are an important goal. Current BioBrick parts are physically composable using a standard assembly method. In this project, we will combinatorially assemble promoters and ribosome binding sites (RBS) in order to test functional composability. The question that we hope to answer is: does a promoter or RBS part function predictably in different contexts?
We will be assembling DNA systems consisting of a promoter followed by a RBS followed by a reporter. The function of a promoter is to initiate transcription of the DNA into RNA. The function of the RBS is to initiate translation of the reporter RNA into protein. Promoters and RBSs can have different strengths and efficiencies. A strong promoter or strong RBS is expected to produce more reporter protein than a weak promoter or weak RBS.
Reporters provide an easy way for us to take a peek at what is happening within a microscopic biological system. In this project, we will use two of the most common reporters: GFP, which makes cells green, and lacZ, which is an enzyme that can make cells different colors. The specific reporter we will be using has the part number E0050 and it consists of both GFP and lacZ.
All parts need a helper construct, known as a plasmid, to be replicated successfully inside a cell.
A miniprep is a way of extracting plasmid DNA from cells. You will be provided with E. coli cells expressing the plasmid pSB4A5. pSB4A5 will be the destination plasmid used for all assemblies. It contains an ampicillin resistance gene (hence the "A" in its name).
We will be using the following protocol: Miniprep/Qiagen_kit
The polymerase chain reaction (PCR) is used for DNA amplification. In PCR, two short DNA fragments, known as primers, are required. The region between the two primers is selectively amplified. Besides amplification of existing DNA, PCR can also be used to add extra sequence to the ends of the ampified DNA.
In this project, you will be amplifying E0050 using a "forward" primer that contains a specific RBS. Thus, before the PCR you will begin with tiny amounts of E0050. After the PCR, you will have a lot of an RBS.E0050 construct.
A restriction digest is used to cut DNA at specific sequences to leave sticky ends. We will be cutting the RBS.E0050 constructs with XbaI and PstI. The destination plasmid pSB4A5 will be cut with EcoRI and PstI.
Many reactions such as PCR and the restriction digest require some form of cleanup after the reaction. We will use agarose gel electrophoresis and spin columns to purify reactions.
DNA ligation is used to join together pieces of DNA that have matching sticky ends. You will be given promoters that have sticky ends that match EcoRI and XbaI. You will do a three way ligation of:
- pSB4A5 (EcoRI and PstI cut)
- promoter (EcoRI and SpeI cut) (SpeI sticky end matches XbaI)
- RBS.E0050 (XbaI and PstI cut)
The result will be a single circular piece of DNA containing pSB4A5.promoter.RBS.E0050.
After ligation, you will transform the circular plasmid DNA into E. coli cells. You will be provided with chemically competent cells that have been prepared to take up DNA.
Transformation is extremely inefficient. The vast majority of the transformed cells will not have taken up the desired DNA. We will place the transformed cells on the antibiotic ampicillin which will kill off all cells that did not take up our plasmid. Furthermore, we will add some extra chemicals in the plate that react with lacZ to make the cells turn blue. Thus, we will select the bluest and greenest cells as the most likely to have the correct construct.
To confirm for sure we have the right DNA sequence, we will take the DNA from a single bacterial colony and send it out for DNA sequencing.
As our final constructs will contain both GFP and lacZ, we could in theory choose to measure either (or both) reporters. For this project, we will be measuring lacZ (also known as β-galactosidase). Different people will be working on constructing and characterizing different combinations of promoters and RBS. At the end, we will try to answer the question, "is this set of parts functionally composable?"