Part:BBa_K1383000

BBa_K1383000 (mCherry- Native RBS)

Usage and Biology

The ribosome binding sites (RBS) of archaea are not well characterized. By creating and characterizing a library of RBS sequences, researchers will be able to express proteins of interest at variable levels of expression in Methanococcus maripaludis. Ribosome binding sites are typically 6-7 base pair sequences on a transcript that is complementary to the 3’ end of the 16S rRNA. After binding of the RBS to the ribosome, translation will be initiated. An RBS with higher affinity for the ribosome will result in higher rate of translation, and inversely, an RBS with lower affinity will result in lower rate of translation.

Characterization

Characterization of the RBS sequences was accomplished using the red fluorescent protein, mCherry as a reporter. Qualitative analysis was evaluated by visualization of the RFP and quantitative analysis was completed through use of a plate reader for reading fluorescence.

Improving the Construct and Characterization of Fluorescent Reporters for use in Methanogens

Last year, the UGA-Georgia team submitted the part BBa_K1138002, named pAW50-mCherry. This construct, designed for use as a fluorescent reporter in methanogens, had a few issues. First, the part was not 100% biobrick compatible due to an internal restriction site in the mCherry gene. The characterization of the part was also inconsistent among samples, and overall unreproducible. To improve upon the previous part, we designed pMEV4-mCherry (BBa_K1383000, figure 1). The primary differences between this construct and the pAW50-mCherry of 2013 is that pMEV4-mCherry does not contain the internal restriction site and the selective resistance gene against puromycin, our antibiotic for use in M. maripaludis, has an independent promoter. Therefore, pMEV4-mCherry is 100% biobrick compatible, and can more reliably function under increased selective pressure. Improvements on characterization are elaborated more below.

UGA-Georgia 2014 Figure 1: The pMEV4-mCherry vector contains a region immediately upstream of the RFP, mCherry, labeled RBS 1-39. This site is where the native RBS and 38 variants thereof will be inserted.

UGA-Georgia 2014 Figure 2: Specific sequence for the 'native' RBS. The region labeled 'Linker' is a few random base pairs that provide the ability to hybridize a gene of interest to the RFP reporter, mCherry. The figure is not drawn to scale.

UGA-Georgia 2014 Figure 3: Visualization of mCherry after 20h of oxygen exposure. The part described on this page, the native RBS, is shown in the middle column.

In an effort to expand synthetic biology research for Archaea, we have developed protein expression tools to facilitate fluorescence mediated detection of proteins. For our 2014 project we present 3 new BioBrick parts that iGEMers can use readily. All three of our parts are BioBrick compatible. Specifically, we constructed tools consisting of native/ synthetic Methanococcus RBS site(s) upstream of a gene encoding red fluorescent protein-mCherry. Prior to cloning the mCherry gene was codon optimized for expression in Methanoccocus (also, ensuring BioBrick compatibility in the design considerations). The BioBrick part- BBa_K1383000 consists of the native Methanococcus RBS site upstream of the mCherry gene. This fragment was inserted into pSB1C3 plasmid backbone using EcoRI and PstI restriction enzymes.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]