"

From 2012.igem.org

• 
• Home
• Team
  • Team
  • Gallery
  • Team Profile
• Project
  • Project description
  • Overview
  • YES gates
  • Riboswitch (RS)
  • RNA polymerases
  • Reporter system standard
  • The phage
  • Modeling
• Parts
• Notebook
• Safety
• Attributions & Achievements
• Human Practices
  • Articles about us
  • High School lecture
• Sponsors & Thanks

Objective

There are two parts in our system, the Phage and the E. coli. Before we can test them together, we have to make sure that our parts work individually. For that purpose, we allocated two team members to create assays that can test the function of our E. coli parts, the inducible RNA polymerases, and characterize them.
The cassettes that are used in the plasmids are shown in Figure 1.

Figure 1: Assay Plasmids' cassettes.

Work plan

We intend to create two colored assays that can indicate and quantify the expression of the different polymerases from our plasmids. Each assay gene will be cloned after each of the RNA polymerase promoters, so that the expression of the polymerases will be linked to the color developed. Before using them with the designated RNA polymerase plasmid, we intend to characterize them by using different inducer concentrations and measuring the absorbance.
Main Parts:

1. Alkaline Phosphatase Assay: In E. coli, alkaline phosphatase (ALP) is encoded by the phoA gene. The wild type phoA gene from Citrobacter has a signal sequence directing export of alkaline phosphatase into the periplasm, where it is active. In this protocol, the colorless substrate p-nitrophenyl phosphate (PNPP) is hydrolyzed by ALP, producing nitrophenol, a yellow color product, which is quantified by an assay at a maximum absorbance 420 nm.
2. xyIE Assay: The xylE gene from Pseudomonas putida TOL pWW0. This gene encodes the enzyme catechol-2,3-dioxygenase (metapyrocatechase), which converts catechol to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde. This is a useful reporter gene; colonies or broths expressing active XylE, in the presence of oxygen, will rapidly convert catechol, a cheap colorless substrate, to a bright yellow compound with an absorbance maximum around 380 nm.
3. Promoters: In order to check as many different polymerases to see with which set of enzymes we achieve minimum cross-reactivity, we worked with 5 polymerases. Both the polymerases and their matching promoters were acquired from Christopher A. Voigt1. He sent us the four RNA polymerases promoters: pT7*,p T3, pK1F, and pN4 with a T7 terminator at the end. These four plasmids will also function as our backbone plasmids, and to them we'll clone the assay genes along with their RBS. The Sp6 promoter was added via PCR over the T7 promoter.
4. Controls:
1. Positive- In order to see whether our assay's genes, phoA and xyIE, work properly and generate a colored output, we created a positive control. The positive control will consist of a Tetracycline repressible promoter (pTetO) before each of the assay's genes. In the absence of an inducer, TetR binds to tetO and prevents transcription. It can be turned on when an inducer, such as tetracycline, binds to TetR and causes a conformation change that prevents TetR from remaining bound to the operator. When the operator site is not bound, the activity of the promoter is restored. Unfortunately, we don't have a bacterium strain with the repressor we plan to use the pTetO as a constitutive promoter.
2. Negative: We have two kinds of negative controls: The first one is to see whether we get any absorbance without the presence of the assay parts, promoter + assay gene. The second one is to measure the leakage of the polymerase promoters. For that reason, we will measure the absorbance of the promoter plasmids without the polymerase genes. 


Figure 2: an illustration of the pET system as designed originally [A] and our own pET as used in our experiment [B]
5. pET Expression System: In order to characterize our assay, we intend to use the pET expression system. The host cell for the pET expression system is a bacteria which has been genetically engineered to incorporate the gene for T7 RNA polymerase, the lac promoter and the lac operator in its genome (BL21 strain). When lactose or a molecule similar to lactose is present inside the cell, transcription of the T7 RNA polymerase is activated. Therefore, we have a similar system to the T7 RNA polymerase plasmid that we intended to create in the first place. By transformation of our pT7 test plasmid to the engineered bacteria with the endogenous T7 polymerase gene, we can characterize our pT7 test plasmid. By adding rising concentrations of the inducer, IPTG, and measuring the absorbance for each one, we can deduce whether we have a rising concentration of our assay genes, phoA and xyIE. However, this only works in bulk or culture assay and not single cell assays, since in single cells the lac system is on/off. Meaning that at low concentrations of IPTG some cells will turn on, while the rest will be off. An illustration of the normal pET system and our "made" system is shown in Figure 2.