Part:BBa_K2957001

IL-8 (CXCL-8) with FLAG Tag

Contents: IL-8 (CXCL-8), FLAG Tag, with Spec/Strep resistance

What is it?: A Basic part; in this particular case we call it a pL0-G (through Modular Cloning Golden Gate Type II Assembly)

What does it do?: It codes for the chemokine, IL-8 (CXCL8), which is involved in an inflammatory response. It was important for our project in particular because of its involvement in the immune system and is known to create neutrophil swarming. It has a Flag tag attached.

How to use it?: You can use this part for Type IIS Cloning. It has BsaI cut sites that allow for this. With other Basic parts like pL0-P (promoter/regulatory sites), pL0-T (terminator part), pL0-5.2/pL0-3 (Inert pieces that form the UTR regions around the genetic circuit), you can use this part to make a composite part. So the pL0's are placed in a given backbone depending on the kind of basic part it is. In this case, we have the CDS/gene part which is in a pL0-G backbone. See more on the Parts pages on our MIT iGEM 2019 wiki: https://2019.igem.org/Team:MIT/Parts. It also has a Flag tag attached to it.

Supplementary Information of IL-8/CXCL-8 by WHU-China 2020

Usage and Biology

Chemokines are a family of small chemotactic cytokines, and the name is derived from their ability to induce directed chemotaxis in nearby responsive cells. All chemokines possess a number of conserved cysteine residues involved in intramolecular disulfide bond formation. Some chemokines can be induced during an immune response to recruit cells of the immune system to a site of infection, while others are considered homeostatic and are involved in controlling the migration of cells during normal processes of tissue maintenance or development. Chemokines are found in all vertebrates, some viruses and some bacteria.

IL-8 is a chemotactic factor that attracts neutrophils, basophils, and T-cells, but not monocytes. It is also involved in neutrophil activation. It is released from several cell types in response to an inflammatory stimulus. IL-8(6-77) has a 5-10-fold higher activity on neutrophil activation, IL-8(5-77) has increased activity on neutrophil activation and IL-8(7-77) has a higher affinity to receptors CXCR1 and CXCR2 as compared to IL-8(1-77), respectively.

[1]Chemokine activity definition [2]UniProtKB - P10145 (IL8_HUMAN)

Results

We used the Transwell experiment(chamber diameter: 6.5 mm, Pore diameter of PC membrane: 8 μm, 48 pores/board) combined with flow cytometry to verify the chemotactic ability of this chemokine on the THP1 cell line.(Fig.1)

Figure 1 Chemotaxis of CXCL8 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l. (n=3)

Besides, we also tested the activity of six other chemokines.(Fig.2)

Figure 2 Chemotaxis index(CI) of 7 chemokines to THP1.(n=3)

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 244
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]