Part:BBa_K733008

lytC + linker + FLAG�

Purpose & Intended Function

Our project seeks to design recombinant bacteria that specifically target and suppress the growth of colorectal carcinoma cells in a controllable way. The binding module of our biological system requires expression of a phage display peptide (RPMrel) on the surface of Bacillus subtilis, the organism we have chosen as our carcinoma suppression vector. We chose to use Imperial College London’s 2010 team’s LytC protein cell wall binding domain as an anchor for chassis surface expression of RPMrel.

This construct positions the FLAG™ peptide where the RPMrel peptide would otherwise be. Tagging LytC with the versatile FLAG™ peptide provides a range of options for characterizing the expression of LytC. Being able to quantify the number of recombinant cell wall binding domains on the chassis surface would help us provide data linking expression quantity and binding ability. Recombinant B. subtilis transformed for expression of this construct can be tested using immunofluorescence and affinity chromatography to, respectively, localize the protein within the cellular environment and purify the protein for quantification.

Subpart Description

LytC (hydrolase cell wall binding domain) 1-954bp region of the 1491bp coding sequence of the LytC protein. Isolated and submitted as a BioBrick by Imperial College London’s 2010 team (details here).

(EAAAK)n type helical linker Stiff, long helical linker designed to separate fusion proteins with minimal disturbance to the function of both proteins. Features distinct 6 amino acid short linker sequence (SRGSRA) at N-terminal. This sequence was included in construction of the recombinant fusion protein LytC - 3xFLAG by [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC262103/pdf/0628.pdf Yamamoto et al (2003)] to allow localization of the LytC protein on the cell wall.

FLAG™ peptide 8 amino acid peptide tag used in attachment to the protein-of-interest to facilitate multiple assays. Assays include affinity chromatography (recombinant protein purification), immunofluorescence (protein localization), and protein electrophoresis (protein detection size studies). FLAG™ is a registered trademark of Sigma-Aldrich Corp. Their product website can be accessed [http://www.sigmaaldrich.com/life-science/proteomics/recombinant-protein-expression/purification-detection/flag-system.html here].

Sequence & 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]

Graphic

Functional Parameters: Austin_UTexas

BBa_K733008 parameters

Burden Imposed by this Part:

Burden Value: -1.5 ± 5.0%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.