Stanford/BIOE44:Module 1:Day4

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Module 1: Day 4

Results

Troubleshooting The Results

Introduction to Arsenic Detoxification Genes in Bacteria

Arsenic is a well known environmental toxicant. For the purpose of this lab class you should be aware that arsenic is often found in one of two oxidation states: arsenite (+3) and arsenate (+5). Arsenic acts as a toxicant through two principal routes. First, arsenate (AsO4), which is chemically similar to phosphate (PO4), can enter through a cell’s phosphate transporters embedded in the cell membrane. In the cell, it can interfere with oxidative phosphorylation. Furthermore, arsenite (AsO3) inhibits crucial cellular enzymes, including the crucial pyruvate dehydrogenase at the core of glucose metabolism (Oremland 2003).

A diverse array of bacteria have evolved mechanisms for detoxification. A well characterized example is the ars operon. Fortunately for you, this genetic system is present in many lab strains of E.coli. While more complex ars operon’s exist, the simplest is composed of three main elements: arsB, arsC, and arsR.

They encode the following proteins

  1. ArsB –an arsenite efflux pump.
  2. ArsC – an enzyme catalyzing the reduction of arsenate to arsenite so that it can be excreted by arsenite transporters.
  3. ArsR – a repressor protein that binds an operon upstream of the ars genes. It provides an example of a negative autoregulation in the absence of arsenic induction.

For more information on diversity of bacteria and archaea that incorporate arsenic into their metabolism, see: Oremland, R. S. and J. F. Stolz (2003). "The ecology of arsenic." Science 300(5621): 939-944.

Building a New BioBrick Part

The part you are going to design is arsR gene and upstream regulation sequence. Recall that the part includes the nucleotide sequence for the arsR gene that encodes the ArsR protein. The ArsR protein is a repressor protein that binds to the operator sequence upstream of the arsR gene. In this way arsR is autoregulated. That is, the expression of arsR turns off the transcription of more arsR mRNA under normal conditions. However, when arsenic is present, the metal binds to the ArsR protein and prevents it from binding to the operator allowing transcription of both the arsR gene and the genes immediately downstream.

The part you are going to rebuild is Part: BBa_J33201 designed and added to the registry by the Edinburgh 2006 iGem team. You can view the part at [[1]]


You could order the part from the registry, but for this class you need to make it from scratch. Indeed the Edinburgh team had could not rely on a catalog. How might you go about this? You should propose two distinct ways to recreate this part.One way can require tools not at your disposal. Another must be possible with the equipment you see in the lab.

  1. Where might you find this genetic material?
  2. How would you be able to get it out of a genome and into a plasmid?
  3. Most importantly, how could you convert a gene to an easy to use biobrick part?

Brainstorm with your classmates and if you need help ask one of the TAs for some hints. Once you have proposed a way to build the part, come find a TA to get a sheet with some further guidelines.

  • Please read:
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