Part:BBa_K3111202

DARPin929_mScarlet_StrepII

This part encodes DARPin929_mScarlet. The use of this part enabled us to study the binding and uptake of DARPin929 by conducting cell culture studies on SK-BR-3 HER2+ cells. The DARPin is flanked by red fluorescent protein mScarlet on the C-terminus which allowed visualisation of binding under confocal microscopy and a StrepII-tag downstream of this fusion, which allows for column-based purification of the expressed protein. It is expressed under a T7 promoter and a strong RBS.

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 575
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1147
  
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental Results

DNA Analysis and Cloning

Figure 1: Test digest of BBa_K3111202 within a pSB1C3 plasmid; a-c indicate repeats of different colonies containing the same plasmid cut with BamHI and XbaI type II restriction enzymes.

We started by cloning BBa_K3111201 into pSB1C3 vector. In order to investigate whether the ligation was successful, we picked 4 colonies from the plate containing the transformed DΗ5α, grown into 5 mL cultures, miniprepped and conducted a test digest with restriction enzymes BamHI and XbaI.

We expected bands at 2976 bp and 580 bp and those were obtained only for colony B as observed in Figure 1. Thus, the miniprepped plasmid obtained from that colony was then transformed into BL21 (DE3) into order to proceed with 50 mL cultures to express the protein.

Day 1

Different batches of BL21 (DE3) competent cells were transformed with pSB1C3 plasmids containing BBa_K3111202 sequence coding for mScarlet + DARPin fusion protein. Transformed cells were grown in LB agar plates containing chloramphenicol and glucose. Plates were incubated at 37°C overnight.

Day 2

Transformed colonies containing pSB1C3_ BBa_K3111202 were used to prepare overnight starter cultures containing a total of 5 mL LB broth and chloramphenicol (5 μL). Cultures were incubated at 37°C overnight.

Day 3

A 50 mL scale-up cultures was prepared from a single starter culture containing cells carrying pSB1C3 + BBa_K3111202. The culture was incubated at 37°C until it reached an OD of 0.6. Once they reached OD 0.6, the cultures were induced by addition of 400 μΜ IPTG. The cultures were left to grow again overnight at 37 °C.

Day 4

The culture was collected and transferred into a 50 mL falcon tube. It was spun for 10 minutes at 5000 rpm in order to pellet the cells. Then the supernatant was discarded and the pellet frozen at -80 °C.