Part:BBa_K2968000

GcvB sRNA with BBa_J23100 promoter with no target binding region

This part has a promoter and sRNA GcvB scaffold but instead of its target region it has terminator and is used as a negative control for experiments to test functional GcvB sRNA construct.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Team KCL_UK 2019

Part BBa_K2968000 is an improvement of the part BBa_K2595000. As shown in figures 1 and 2 part BBa_K2968000 additionally this new part has BBa_J23100 promoter part, slightly modified BaeI linker 1 region and double transcriptional terminator part BBa_B0015 in place of the single terminator. These modifications enable us not only to speed up the creation of the functional sRNA but also to better inhibit an undesirable expression of the sRNA scaffold when we used part BBa_K2968000 as a negative control in our experiments. Total inhibition of the sRNA scaffold expression for the negative control experiment is necessary as our preliminary laboratory experiments have indicated that for some reasons some sRNA scaffolds when expressed from a strong promoter inhibit bacterial growth. To add a target binding region, new part BBa_K2968000 also allows teams to use two separate methods, either to digest this part with BaeI restriction enzyme or to use a site directed mutagenesis protocol. To confirm that BBa_K2968000 part is an improvement of the BBa_K2595000 we used BBa_K2968000 part as a template in a site directed mutagenesis experiment and creates in one day a functional sRNA part BBa_K2968001 https://parts.igem.org/Part:BBa_K2968001. We tested this part together with the pSB4K5_BBa_K608011 reporter plasmid and have shown that the newly created sRNA completely inhibits translation of the GFP gene.

Figure 1. Annotated sequence of the BBa_K2595000 part

Figure 2. Annotated sequence of the BBa_K2968000 part