Part:BBa_K1137000

M. Smegmatis SirA

This biobrick encodes a ferredoxin-dependent sulfite reductase SirA, which reduces sulfite to hydrogen sulfide in the cysteine metabolism pathway. Due to the sensitivity of sulfur contain amino acids to oxidative stress; they must constantly be replenished in mycobacteria while inside the phagosome. Thus SirA is one of the few genes that is upregulated even in latent mycobacterial infections. Additionally, SirA differs from E. coli and human sulfite reductases in its use of ferredoxin as an electron done instead of NADPH. As a result SirA has been previously identified as a drug target candidate for M. tuberculosis infections. SirA reduces sulfite in the following reaction:

(EC=1.8.7.1)

〖SO〗_3^(2- )+6〖FdxA〗_red+6H^+ ↔ H_2 S+6〖FdxA〗_ox+3H_2 O

Thus this gene can be used to provide cysteine in E. coli strains that lack a native CysI gene and allow for growth on minimal media. However, to do so it requires co-expression of biobricks BBa_K1137001 and BBa_K1137002. Even with minimal induction in a duet vector expression system, we were able to obtain growth in M9 media supplemented with glucose as a carbon source when co-expressed with BBa_K1137001 and BBa_K1137002, however the parent strain which lacked these biobricks was unable to grow. M. smegmatis SirA has high homology (about 89%) to SirA from M. tuberculosis.

Growth curves: BL21 (DE3) ΔcysI containing the MycoSIR pathway (MycoSIR E.coli) were grown in liquid minimal media containing Various concentration of IPTG. (A) Replicates of each strain were measured for absorbance in a spectrophotometer every 10 minutes for 14 hours. Growth was observed for the WT BL21 E.coli, (blue), and the MycoSIR E.coli (red). No growth was detected for uninduced MycoSIR E.coli (purple) or for the BL21 (DE3) ΔcysI that did not contain the synthetic pathway (Orange) . (B) Mean Final ODs of all replicates, measured after 14 hours of growth. Growth was detected in zmSIR E.coli and WT BL21 but not in uninduced zmSIR strain.

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 152
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 711
  Illegal AgeI site found at 994
  
• 1000
  COMPATIBLE WITH RFC[1000]