Part:BBa_K3558000:Design

Small Heat Shock Protein 22E (sHSP22E)

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 455
  Illegal PstI site found at 461
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 455
  Illegal PstI site found at 461
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 455
  Illegal PstI site found at 461
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 455
  Illegal PstI site found at 461
  
• 1000
  COMPATIBLE WITH RFC[1000]

Plasmid Design and Cloning

Plasmid Design with HSP22E and HSP22F Inserts

DNA sequences for our genes of interest (HSP22E and HSP22F) were obtained from Genbank. Gene constructs for each were designed with the following features: 1. Forward and reverse overhangs were added onto both 5’ and 3’ ends of the gene sequences to enable Gibson assembly. The overhangs were complementary to the pET-19b plasmid backbone.

    a. Fwd: 5’ CGGCTGCTAACAAAGCCCGA 3’
    b. Rev: 5’ CTTTAAGAAGGAGATATACC 3’

2. 6x His-tag and GSG linker - The His-tag consisted of six histidines, which enable protein purification using an immobilised metal affinity chromatography column. The GSG linker allowed for protein folding without interference by the 6xHis-tag.

    a. 5’ GGCTCCGGCGGACATCATCATCATCACCATTAA 3’

As the HSP22 genes were obtained from eukaryotic C. reinhardtii, gene constructs were codon optimised for E. coli using the IDT Codon Optimisation Tool. DNA g-blocks were synthesised by Integrated DNA Technologies (IDT).

Constructs were designed to be inserted into the pET-19b plasmid backbone (figure 1), a standard E. coli protein expression vector. The plasmid possesses an ampicillin resistance gene to allow for selection of successfully transformed colonies. It also utilises the T7 expression system under the control of a modified Lac operon. The LacI gene produces a lac repressor, which binds to the lac operator. This has been placed downstream of the T7 promoter. Only when IPTG is added is the lac repression relieved, allowing for the T7 polymerase to bind to the T7 promoter and begin transcription of the inserted gene. (1) The DE3 strains have been modified with a copy of the phage T7 RNA polymerase gene and thus were chosen as our experimental chassis. This expression system was chosen as it is inducible and the T7 mechanism ensures almost no leakage.

Figure 1: Diagram of pET-10b plasmid with the insert HSP22E. A similar plasmid was designed for the HSP22F insert. Ampicillin resistance gene (AmpR) and Lac repressor (LacI) can be seen alongside their respective promoters. Gene was inserted with designed overhangs in between T7 promoter and terminator. Image produced on Benchling.

Design Notes

The sHSP22E codons were optimised via IDT's Codon Optimization Tool. His-tags were also added to the C-terminal of the sHSP22E sequence for purification purposes in the later stages of the the experiment.

Source

The sHSP22E sequence was retrieved from GenBank. (2)

References

1. Mierendorf RC, Morris BB, Hammer B, Novy RE. Expression and Purification of Recombinant Proteins Using the pET System. In: Rapley R, editor. The Nucleic Acid Protocols Handbook [Internet]. Totowa, NJ: Humana Press; 2000 [cited 2020 Oct 28]. p. 947–77. (Springer Protocols Handbooks).

2. Chlamydomonas reinhardtii strain CC-503 cw92 mt+ CHLREscaffold_1 genom - Nucleotide - NCBI [Internet]. Ncbi.nlm.nih.gov. 2020 [cited 22 October 2020]. Available from: https://www.ncbi.nlm.nih.gov/nuccore/NW_001843471.1?from=5781321&to=5782811&report=genbank