Composite

Part:BBa_K3187001

Designed by: iGEM TU_Darmstadt 2019   Group: iGEM19_TU_Darmstadt   (2019-10-12)
Revision as of 08:06, 15 October 2019 by Franziska (Talk | contribs)


P22 Bacteriophage Coat Protein expression cassette

Coat Protein

name coat protein
base pairs 1293
molecular weight 46,9 kDa
origin bacteriophage P22
parts coat protein, T7Te promoter, lac-operator, rrnb T1 terminator, Strep-tagII
properties Assembly with scaffold protein to a Virus-like particle

Usage and Biology

This part encodes the coat protein (CP) (BBa_K3187017) of the bacteriophage P22 capsid. Importantly, it must not be confused with coat proteins in membrane transport of eukaryotic cells. Coat Protein is an umbrella term for many different proteins, which simplify the transfer of molecules between different compartments that are surrounded by a membrane. [1]
In the natural context of P22, its genetic information is included and protected by the capsid, before it is transferred into the host organism during infection. Bacteriophagic coat proteins have been used for many purposes, for example vaccines or drug delivery. [2]
The P22 coat protein consists of 431 amino acids and its molecular weight is 46,9 kDa. Because it is found in the structural components of viral proteins, it is an important part of Virus-like particles (VLP) as well. Together with the scaffold protein (BBa_K3187021), the proteins assemble to a VLP and build the basis for our modular platform.

The coat proteins (BBa_K3187001) are heterologously expressed in E. coli BL21 (DE3). As backbone the pACYCT2 plasmid is used, containing a T7 promoter, lac-operator and RBS (BBa_K3187029). Moreover the part comprises a C-terminal StrepTagII (BBa_K3187025), a Short Linker (5AA) (BBa_K3187030) and two terminators, T7Te terminator and rrnb T1 terminator (BBa_K3187036).

Methods

Cloning

The sequence of the coat protein ordered from Integrated DNA Technologies (IDT) was inserted in the pACYCT2 backbone. For this purpose, the Gibson asssembly was used. The sequence was verified by sanger sequencing through Microsynth Seqlab.

purification

The heterologous expressed coat protein in E. coli was purified using a GE Healthcare ÄKTA Pure machine which is a machine for FPLC

SDS-Page and Western blot

To verify that the coat protein was heterologous produced, a SDS-Page followed by a Western blot was performed.

Results

Cloning and Expression

The ordered sequence from IDT was cloned into the pACYCT2 plasmid with Gibson assembly and heterologous expressed in E. coli. The accuracy of cloning was controlled via sanger sequencing (Microsynth Seqlab ) and the expression was observed using an SDS-Page and Western blot. The Western blot showed a band corresponding to the size of approximately 46,9 kDa. So, the successful cloning and expression was proven.

Figure 1: Western blot of all produced and purified proteins.

Fig. 1 shows that the CPs were detected with anti-strep antibody. The band of the CP is approximatley found by the 46.9 kDa band. Consequently, the successful cloning and expression was proven.

References

  1. ↑ Juan S. Bonifacino, Jennifer Lippincott-Schwartz, Coat proteins: shaping membranetransport, NATURE REVIEWS MOLECULAR CELLBIOLOGY, May 2013, 4, 409-414 [1]
  2. ↑ Rohovie, Marcus J., Maya Nagasawa, and James R. Swartz. "Virus‐like particles: Next‐generation nanoparticles for targeted therapeutic delivery." Bioengineering & translational medicine 2.1 (2017): 43-57 [2]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1458
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1504


[edit]
Categories
Parameters
None