20.109(F20):M3D1

Introduction

In this module you build on the progress of former 109ers! In previous semesters, students engineered the the metabolism of E. coli in an attempt to increase the production of a commercially relevant product. Specifically, metabolic engineering was used to increase the ethanol yield via manipulating the fermentation pathway native to E. coli cells. Ethanol, or ethyl alcohol, is used as fuel – most commonly as a biofuel additive in gasoline. Current methods for producing ethanol involve the use of agricultural feedstocks and concern is mounting given the effect this may have on food prices and resources. Using E. coli as an ethanol-generating machine is promising; however, the ethanol output of a native cell is very low.

Metabolic engineering refers to the alteration of genetic and/or regulatory circuitry within organisms. The native circuitry involves a series of enzymes that perform biochemical reactions that function to convert raw substrates into products that are required for the organism’s survival. When this native circuitry is altered using metabolic engineering techniques, the goal is to use the host organisms as machines that produce valuable materials in large quantities and at low cost.

Example illustrating pathway to desired product.

Common strategies employed in metabolic engineering are:

• Increasing expression of a gene that encodes an enzyme responsible for a rate-limiting step.
• Inhibiting competing pathways that divert substrate away from the pathway of interest.
• Incorporating genes from other organisms into the host.
• Altering the structure of the enzyme such that yield is increased.

To increase production of a desired product, you can either target proteins that use the substrate to generate alternate products (see A in image on right) or you can target proteins that use the desired product to generate alternate products (see B in image on right). In A the substrate is siphoned away from the reaction that generates the desired product and in B the desired product is used as substrate in a subsequent reaction. By eliminating the proteins that catalyze the reactions that result in alternate products, you can potentially increase production of your desired product.

To manipulate the metabolism of E. coli, you will use the CRISPRi system. Today you will design a guide RNA (gRNA) that specifically targets a gene within the fermentation pathway of E. coli.

Unlike more commonly used CRISPR-based technologies, CRISPRi is used to modulate expression from the genome rather than to modify the genome. This distinction is due to the use of an enzymatically-inactive dCas9 (or ‘dead’ Cas9) protein. Because dCas9 is enzymatically inactive, it is unable to cleave the DNA upon binding to a gRNA/DNA complex. The lack of DNA cleavage results in gene silencing through impeding RNA polymerase binding, transcription factor binding, and/or transcription elongation. The method of repression is largely due to the location of the genome that is targeted. As you may recall from your gRNA design considerations, if you target the promoter of a gene of interest, you can influence transcription by all methods, and if you target a sequence within the gene, you will influence only elongation.

Schematic of CRISPRi preventing transcription by RNA polymerase. Image taken from Qi Lab Homepage.

Protocols

Part 2: Review ethanol metabolism via the E. coli fermentation pathway

Your goal in this module is to increase the production of either ethanol or acetate, two valuable products of the E. coli mixed-acid fermentation pathway. You will complete this task using the CRISPRi system, which targets genes such that transcription of the target gene is decreased, thus resulting in less of the protein encoded by the targeted gene.

Use the fermentation pathway from E. coli (included below) and the example above to answer the following questions with your partner.

1. Which gene(s) might you target to increase the availability of substrate for ethanol production? For acetate production?
2. Which gene(s) might you target to decrease the amount of substrate used to generate products in steps downstream of ethanol production? of acetate production?

Mixed-acid fermentation in E. coli. Image from Forster and Gescher, Front. in Bioeng. and Biotech., 2014.

With your laboratory partner, review Metabolic engineering of Escherichia coli for production of mixed-acid fermentation end products by Forster & Gescher. Using the information within this article and the genes that you identified above, select one gene that you will target in an attempt to increase the production of either ethanol or acetate. Be sure to include notes on your decision and thought process in your laboratory notebook.

Before you continue, note the fermentation product you aim to increase and the gene you will target in the Class Data Page.

PCR amplification

The applications of PCR (polymerase chain reaction) are widespread, from forensics to molecular biology to evolution, but the goal of any PCR is the same: to generate many copies of DNA from a single or a few specific sequence(s) (called the “target” or “template”).

In addition to the target, PCR requires only three components: primers to bind sequence flanking the target, dNTPs to polymerize, and a heat-stable polymerase to carry out the synthesis reaction over and over and over. DNA polymerases require short initating pieces of DNA (or RNA) called primers in order to copy DNA. In PCR amplification, forward and reverse primers that target the non-coding and coding strands of DNA, respectively, are separated by a distance equal to the length of the DNA to be copied. Length is one important design feature. Primers that are too short may lack requisite specificity for the desired sequence, and thus amplify an unrelated sequence. The longer a primer is, the more favorable are its energetics for annealing to the template DNA, due to increased hydrogen bonding. On the other hand, longer primers are more likely to form secondary structures such as hairpins, leading to inefficient template priming. Two other important features are G/C content and placement. Having a G or C base at the end of each primer increases priming efficiency, due to the greater energy of a GC pair compared to an AT pair. The latter decrease the stability of the primer-template complex. Overall G/C content should ideally be 50 +/- 10%, because long stretches of G/C or A/T bases are both difficult to copy. The G/C content also affects the melting temperature. PCR is a three-step process (denature, anneal, extend) and these steps are repeated 20 or more times. After 30 cycles of PCR, there could be as many as a billion copies of the original target sequence.

Based on the numerous applications of PCR, it may seem that the technique has been around forever. In fact it is just over 30 years old. In 1984, Kary Mullis described this technique for amplifying DNA of known or unknown sequence, realizing immediately the significance of his insight.

"Dear Thor!," I exclaimed. I had solved the most annoying problems in DNA chemistry in a single lightening bolt. Abundance and distinction. With two oligonucleotides, DNA polymerase, and the four nucleosidetriphosphates I could make as much of a DNA sequence as I wanted and I could make it on a fragment of a specific size that I could distinguish easily. Somehow, I thought, it had to be an illusion. Otherwise it would change DNA chemistry forever. Otherwise it would make me famous. It was too easy. Someone else would have done it and I would surely have heard of it. We would be doing it all the time. What was I failing to see? "Jennifer, wake up. I've thought of something incredible." --Kary Mullis from his Nobel lecture; December 8, 1993