Part:BBa_K1185001

HBsu-sfGFP

This BioBrick codes for a HBsu protein attached to a superfolded green fluorescent protein (sfGFP). HBsu is a non-specific DNA binding protein that binds to DNA as a homodimer. The HBsu is joined to the sfGFP through ten amino acid flexible linker sequence. This allows the observation of Bacillus subtilis DNA using fluorescence microscopy. The integration strategy that we opted for was to clone in the BioBrick into the Multiple cloning site of pMutin4 backbone. First we attached HindIII restriction sites on 5'end and SacII restriction sites on 3'end of the BioBrick. We then cut the pMutin4 backbone and BioBrick using the previously mentioned restriction enzymes, this allowed us to ligate the plasmid and BioBrick together. We also attached a ~300bp amyE homology region onto the 3'end of the BioBrick after the SacII to allow the single cross over and integration of the whole plasmid into the genome. The pMutin4 plasmid contains a ery+ resistance marker for B.subtilis and amp+ for E.coli and also contains lacI, lacZ and Pspac promoter which is an IPTG induced promoter which regulated the transcription of this BioBrick. An alternative method to use this part would be to clone this BioBrick out and use any Assembly protocol to attach a desired promoter, RBS and antibiotic resistance genes.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 339
  

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)  TTTGGGAGGAGGTGAAAGGCATGAACAAA ... CTGTACAAATGATAAGTAAGTAA  ORF from nucleotide position 21 to 1040 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below) 1  101  201  301  MNKTELINAVAEASELSKKDATKAVDSVFDTILDALKNGDKIQLIGFGNFEVRERSARKGRNPQTGEEIEIPASKVPAFKPGKALKDAVAGKGENLYFQS GGMRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTI SFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDN HYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK*
Sequence features: (with their position in the amino acid sequence, see the list of supported features) TEV cleavage site:  94 to 100
Amino acid composition: Ala (A) 21 (6.2%) Arg (R) 12 (3.5%) Asn (N) 19 (5.6%) Asp (D) 24 (7.1%) Cys (C) 2 (0.6%) Gln (Q) 11 (3.2%) Glu (E) 25 (7.4%) Gly (G) 32 (9.4%) His (H) 10 (2.9%) Ile (I) 17 (5.0%) Leu (L) 27 (7.9%) Lys (K) 32 (9.4%) Met (M) 6 (1.8%) Phe (F) 17 (5.0%) Pro (P) 14 (4.1%) Ser (S) 14 (4.1%) Thr (T) 22 (6.5%) Trp (W) 1 (0.3%) Tyr (Y) 10 (2.9%) Val (V) 24 (7.1%)
Amino acid counting Total number: 340 Positively charged (Arg+Lys): 44 (12.9%) Negatively charged (Asp+Glu): 49 (14.4%) Aromatic (Phe+His+Try+Tyr): 38 (11.2%)	Biochemical parameters Atomic composition: C1677H2637N459O515S8 Molecular mass [Da]: 37725.6 Theoretical pI: 6.09 Extinction coefficient at 280 nm [M-1 cm-1]: 20400 / 20525 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges
Codon usage Organism: E. coli B. subtilis S. cerevisiae A. thaliana P. patens Mammals Codon quality (CAI): excellent (0.83) excellent (0.80) good (0.67) good (0.73) excellent (0.80) good (0.70)
Alignments (obtained from PredictProtein.org)    There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.
Predictions (obtained from PredictProtein.org)
There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment.

HBsu-sfGFP Testing and Characterisation

To test that HBsu-sfGFP (BBa_K1185001)BioBrick works as designed; we transformed Bacillus subtilis str. 168 with the pMUTIN4 plasmid containing our HBsu-sfGFP construct. There were colonies on Plate 1 which is LB + 5ug/ml ery plates containing the transforms B. subtilis with the plasmid containing our constructs as well as plate 2 which act as our positive control. There is no colonies on the negative control plate (Plate 3) suggesting that the colonies on both Plate 1 and Plate 2 are indeed bacteria that has been successfully transformed with the plasmids instead of backgrounds. The results of the experiments are display in Figure 1.

Plate 1: B. subtilis transformed with HBsu-sfGFP plated on LB + ery (5ug/ml) plate.	Plate 2: B. subtilis transformed with pGFPrrnB plated on LB + ery (5ug/ml) plate(positive control).
Plate 3: B. subtilis transformed with water plated on LB plate (viability of cells).	Figure 1. Transformation plates of ''Bacillus subtilis'' with HBsu-sfGFP, pGFPrrnB (as possitive control) and water as control to check the viability of cells.

In order to make sure that the BioBrick had been integrated properly into the endogenous amyE region, the transformants were plated out onto Starch plate. As the BioBrick integrated into the amyE region the cells did not produce amylase thus were unable to digest starch, and when iodine was applied to the cells they did not have a zone of clearance. As can be seen in Figure 2, both B. subtilis with either HBsu-sfGFP or HBsu-RFP constructs don't have any zone of clearance around the colonies in comparison to wild type B. subtilis str.168 and B. subtilis BSB1. The results of this iodine test further confirm that the transformants seen on Plate 1 Figure 1 were indeed colonies with our contruct integrated into the amyE region.

Figure 2. Starch plate showing alpha amylase activity. The ''B. subtilis'' 168 HBsu-GFP and HBsu-RFP colonies have no zones of clearance indicating that our HBsu-sfGFP and HBsu-RFP have integrated into the B. subtilis chromosome via the ''amyE'' region stopping starch digestion.

To gain evidence that our BioBrick is actually binding to DNA we stained the genome of B. subtilis cells transformed with our BioBrick with DAPI. The binds DNA and emits a blue fluorescence at 465nm. As can be seen in figure 3, the localisation of the DAPI coinsides with the green fluorescence emitted by HBsu-sfGFP at 560nm, suggesting that the HBsu-RFP is binding to the B. subtilis chromosome.

Figure 3. Left hand image shows B. subtilis Phase contrast image, the central shows the bacteria expressing HBsu-sfGFP and the right corner shows fluoresecense with DAPI staining. The HBsu-sfGFP and DAPI fluorescence shows same localisation in signal, indicating our BioBrick actually binds to DNA.

• Figure 4

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  10 minutes	30 minutes
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  240 minutes	Figure 4. Negative control: Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.

• Figure 5

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  10 minutes	30 minutes
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  240 minutes	Figure 5. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.05mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.

• Figure 6

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  10 minutes	30 minutes
  60 minutes	120 minutes
  240 minutes	Figure 6. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.1mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.

• Figure 7

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  10 minutes	30 minutes
  60 minutes	120 minutes
  240 minutes	Figure 7. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.2mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.

• Figure 8

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  10 minutes	30 minutes
  60 minutes	120 minutes
  240 minutes	Figure 8. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.4mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.

• Figure 9

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  10 minutes	30 minutes
  60 minutes	120 minutes
  240 minutes	Figure 9. Fluorescence microscopy showing how sfGFP expession changes with time on addition of 0.8mM IPTG in B.subtilis containing our HBsu-sfGFP BioBrick.

Figure 10. Chart showing the level of GFP expression by B. subtilis at time intervals after the addition of IPTG in different concentrations.

Figure 10 shows the levels of sfGFP expression in B.subtilis transformed with the HBsu-sfGFP BioBrick, with varying concentrations of IPTG, quantitatively. It can be seen that 10 minutes incubation, even with high levels of IPTG (0.8mM), is an inadequate time span to elicit visible HBsu-sfGFP expression. After 30minutes there is a marked increase in expression of HBsu-sfGFP in cultures exposed to 0.8mM IPTG. Cultures exposed to higher levels of IPTG typically exhibit higher levels of expression in the same time frame than those exposed to lower levels of IPTG. Levels of IPTG below 0.1mM have not been shown to induce HBsu-sfGFP expression. Table 1 shows this data numerically.

Table 1. Table showing the level of GFP expression by B. subtilis at time intervals after the addition of IPTG in different concentrations.

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.]

[http://www.ncbi.nlm.nih.gov/pubmed/1902464 Micka, B., Groch, N., Heinemann, U. & Marahiel , M., 1991. Molecular cloning, nucleotide sequence, and characterization of the Bacillus subtilis gene encoding the DNA binding protein HBsu. Journal of Bacteriology, May;173(10), pp. 3191 -3198.]

[http://www.ncbi.nlm.nih.gov/pubmed/1382620 Micka, B. & Marahiel, M., 1992. The DNA-binding protein HBsu is essential for normal growth and development in Bacillus subtilis. Biochimie, 74(7-8), pp. 641-650.]

[http://www.ncbi.nlm.nih.gov/pubmed/10715001 Ross , M. & Setlow, P., 2000. The Bacillus subtilis HBsu protein modifies the effects of alpha/beta-type, small acid-soluble spore proteins on DNA.. Journal of Bacteriology, 182(7), pp. 1942-1948.]