Contents 1 HBsu-xFP Fusion 1.1 Purpose and Justification 1.2 Assembly 1.2.1 HBsu-(x)FP 1.3 Integration 1.4 Testing and Characterisation 1.5 References

HBsu-xFP Fusion

Purpose and Justification

Bacillus subtilis contains HBsu, a non-specific DNA binding protein which is a homologue to eukaryotic histones. This protein is encoded by the gene hbs gene which is 263bp in length and involved not only in DNA binding but also SRP, DNA repair, HR, and pre-secretory protein translocation [http://www.ncbi.nlm.nih.gov/pubmed/10224127 (Kouji, et al., 1999)]. It binds to DNA by forming homodimer.

We conjugated the hbs gene with red fluorescent protein (RFP/GFP) in order to fluorescently tag the bacterial chromosome. We produced [http://parts.igem.org/Part:BBa_K1185001 BBa_K1185001] which codes for a HBsu-sfGFP conjugate and [http://parts.igem.org/Part:BBa_K1185002 BBa_K1185002] which coding for a HBsu-RFP conjugate. Each bacterium was transformed with only one or the other. The expression of these BioBricks was regulated through an IPTG induce promoter (Pspac).

When one bacteria containing HBsu-RFP and another containing HBsu-sfGFP are fused together, we can view the genomic shuffling via fluorescent microscopy. These BioBrick is the fusion of two L-form B. subtilis, and aims to improve existing phenotype via genome shuffling. This can be visualised through fluorescent microscopy, if a colour mosaic is formed we can confidently claim that recombination has occurred.

Assembly

HBsu-(x)FP

BioBrick encodes a fusion protein HBsu-(x)FP protein by 10 amino acids flexible linker (BBa_K105012).

The first version of the BioBrick have superfolder GFP (BBa_E0040)conjugated to the HBsu.

The second version of the BioBrick have RFP (BBa_E1010) conjugated to the HBsu.

Integration

Testing and Characterisation

References

[http://www.ncbi.nlm.nih.gov/pubmed/10224127 Kouji, N., Shou-ichi, Y., Takao, Y. & Kunio , Y., 1999. Bacillus subtilis Histone-like Protein, HBsu, Is an Integral Component of a SRP-like Particle That Can Bind the Alu Domain of Small Cytoplasmic RNA. The Journal of Biological Chemistry , Volume 274, pp. 13569-13576.]