Part:BBa_K3182000

pT7-CBDcipA-AsPink

A cellulose binding domain (CBD) from Clostridium thermocellum which can be used to purify or attach proteins to cellulose. The part also has a flexible GS-linker with a thrombin site added at the end, clevage with thrombin will add one glycine and one serine to the N-terminal of the fusion protein.

The part has a AsPink (Codon opt. BBa_K1033933) fused to the CBD to ensure easy trackable expression and characterization ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946454 Liljeruhm et al. 2018]) of the CBD. The part has a very strong expression with a T7 promotor (BBa_I719005) as well as a 5'-UTR (BBa_K1758100) region which has been shown to further increase expression in E. coli (BBa_K1758106), ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]).

Figure 1. E. coli BL21 cells expressing this biobrick, incubated for 16 hours at 16°C at 80 rpm in 1 litre of LB-miller.

Figure 2. Lysated (via sonication) BL21s which are expressing the biobrick. It is lysate from the culture above in figure 1. Incubated for 16 hours at 16°C at 80 rpm in 1 litre of LB-miller.

Figure 3. Centrifuged lysate of BL21 culture which express the biobrick. It is the same culture as figure 1 and 2. A pellet of non-lysated bacteria can be observed.

Potential Usages

AsPink can for example be used as a reporter for CBD binding ability, track purification of the CBD or report linker breakage. By including a BamHI site on the linker; the asPink can be switched with another protein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 580
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]