Part:BBa_K1481003

sfGFP with R4-tag under arabinose promoter

This part generates a monomeric red fluorescent protein with his-tag for easy protein purification and R4 degradation tag comprising of four consecutive arginine codons. It is controlled by a pBAD arabinose-inducible promoter. It also contains a coding sequence for the AraC protein, crucial for repression of pBAD in absence of arabinose.

Contribution

Group: UAM_Poznan 2016
Author: Daria Niewiadomska

We have tested and slightly improved Ba_K1481003 biobrick provided to iGEM in 2014 by Poznan_Bioinf team, named "sfGFP with R4-tag under arabinose promoter" Firstly we have checked if four rare arginine codons added to the 3' end of GFP reading frame will influence its translation compared to sfGFP reading frame under identical promoter with an identical 5’UTR - biobrick: Ba_K1481002 provided by Poznan_Bioinf as well. Surprisingly we have found that there is no obvious difference in expression of both genes. It was difficult to believe this because many people optimize reading frames to be expressed in E. coli by removal rare codons especially those for arginine, namely AGG and AGA. There are also available special E. coli strains like BL21 Codon Plus [Rosano GL, Ceccarelli, 2009], which encode additional tRNAs and recognize these codons, constructed to enhance the expression of “difficult” ORFs containing rare codons. To be sure that this what we see is not something obtained just by chance, we have extended this tag to eight consecutive arginine residues.

Our construct contains the synthetic sequence coding for sfGFP [Pedelacq JD, 2006] to which we added poly-histidine tag at N-terminus of coding sequence and eight arginine residues at its C-terminus, encoded by rare AGA and AGG codons (Fig. 1.). The expression of this gene is regulated by arabinose promoter (AraC-pBAD)- a tightly controlled and adjustable promoter used in pBAD expression vectors (Invitrogen, Thermo-Fischer).

Fig. 1. The scheme of the biobrick: BBa_K2014007.

In order to check to what extent eight rare arginine codons (AGA, AGG) at the 3’-end of a coding sequence influence the translation we measured the fluorescence of sfGFP_8R reporter during 6h culture of E.coli DH5α in three different media (SB/PKB, LB and M9 minimal medium, see methods section) supplemented with 0,4% of arabinose (Fig. 2, 3, 4). The results were referred to sfGFP (Ba_K1481002) and to sfGFP_Worst (BBa_K2014006). We also decided to compare the fluorescence of sfGFP_8R with another construct sfGFP_TagX. Instead of arginine residues, sfGFP_TagX has degradation tag (a decapeptide sequence: ANDENYALAA), which is added to defective mRNAs devoid of stop codons, by translation of tmRNA which binds to the vacant A-site on a stalled ribosome and directs the truncated protein to proteolysis [Hayes et al, 2001]. The results we obtained indicate that either the 8-arginine tag or degradation tag on C-terminus of coding sequence seems not to be detrimental for recombinant protein production in our system.

Fig. 2. Comparison of four different variants of sfGFP ORFs in E.coli DH5α in M9 minimal medium supplemented with 0,4% of arabinose for 6h at 37°C.

Fig. 3. Comparison of four different variants of sfGFP ORFs in E.coli DH5α in LB medium supplemented with 0,4% of arabinose for 6h at 37°C.

Fig. 4. Comparison of four different variants of sfGFP ORFs in E.coli DH5α in SB/PKB medium supplemented with 0,4% of arabinose for 6h at 37°C.

After bacteria lysis (See [http://2016.igem.org/Team:UAM_Poznan/Experiments Methods]) we observed a substantial fraction of sfGFP_8R still remained in the pellet (Fig. 5). We presume that the added arginine residues at the C-terminus of coding sequence promotes permanent bonding with other structures in cell due to charge–charge interactions [Vondrášek J et al, 2009]. That can be the reason why graphs show a slight decrease in biosynthesis from sfGFP_8R ORF.

Fig. 5. Photograph presenting pellets before lysis (left) and pellets with supernatants after lysis (right) (See: Methods) after 4th hour of culturing E.coli cells in SB/PKB medium with 0,4% of arabinose. White arrows point sfGFP still remained in pellet after lysis, while sfGFP and sfGFP-tagX are fully released from E. coli cells.

Names’ abbreviations:
sfGFP- Ba_K1481002
sfGFP_W- BBaK2014006
sfGFP_8R- BBa_K2014007

REFERENCES
1. GL Rosano and EA Ceccarelli. (2009) Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microbial Cell Factories 2009, 8:41.
2. Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS. (2006) Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol. 2006 Sep;24(9):1170.
3. Hayes CS, Bose B and Sauer RT. (2001) Stop codons preceded by rare arginine codons are efficient determinants of SsrA tagging in Escherichia coli. Proc Natl Acad Sci U S A. 19; 99(6):3440-5.
4. Vondrášek J, Mason PE, Heyda J, Collins KD and Jungwirth P. (2009) The Molecular Origin of Like-Charge Arginine−Arginine Pairing in Water. J. Phys. Chem. B, 2009, 113 (27), pp 9041–9045

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 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  Illegal SapI.rc site found at 1260