Part:BBa_K2968005

sRNA Spot42 targeting BBa_B0034 and BBa_E0040 start codon

This sRNA has promoter target binding region for BBa_B0034 and BBa_E0040 start codon

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

KCL iGEM 2019

BBa_K2968005

To generate functional sRNA molecule with the secondary structure shown in figure 1 our team has created this composite part consisting of three basic parts: BBa_J23100 promoter, BBa_K2968014 mRNA target binding region and BBa_K2968012 Spot42 sRNA scaffold as shown in figure 2.

Figure 1. BBa_K2968005 expressing sRNA secondary structure predicted using http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi server. The mRNA target binding region is highlighted in pink and the sRNA scaffold in teal.

Figure 2. Details of the BBa_K2968005 sRNA construct design

The BBa_J23100 promoter is a strong promoter from the constitutive promoter family designed by John Anderson, iGEM2006_Berkeley. BBa_K2968014 part is a complementary part to the BBa_K608010 that has BBa_B0034 Ribosome Binding Site (RBS) and BBa_E0040 GFP gene part as shown in figure 3. The Spot42 scaffold is the 51 bp region of the E.coli sRNA.

Figure 3. Spot42 sRNA construct target binding region to the GFP mRNA.

To validate our BBa_K2968005 composite part, we transformed Xl1Blue E.coli cells with the pSB1C3 plasmid harbouring this part together with the reporter GFP plasmid pSB4K5 containing BBa_K608010 part. Positive colonies were selected on the LB agar plates containing Kanamycin (15 ug/ml) and Chloramphenicol (34 ug/ml). Example of one of these colonies streaked on the agar plate is shown in figure 4 panel E. Visual analysis indicates that the sRNA expressed from the BBa_K2968005 part inhibits GFP reporter molecule expression as compare to the pane D where target binding region in the sRNA molecule was substituted with the transcription terminator regen. This result confirms that our part BBa_K2968005 is functioning as initially expected by our team.

Figure 4. Analysis of the function of the Spot42 based sRNA in XL1Blue E.coli on LB agar plate under fluorescent light. Panel A. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968003; Panel B. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968004; Panel C. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968004; Panel D. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968003; Panel E. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968005; Panel F. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968005

To further validate our part, a single colony from the plate was inoculated into 10 ml LB media containing Kanamycin (15 ug/ml) and Chloramphenicol (34 ug/ml) and incubated overnight in a shaking incubator at 37 oC with shaking 200 rpm. After approximately 16 h of incubation E.coli cultures were diluted 1/10 with fresh 10 ml LB media containing both antibiotics in 20 ml universal bottle. Each experiment was performed in duplicate. At this point 500 ul of the culture was collected into 1.5 ml centrifuge tube, labelled 0h incubation and stored on ice. The rest of the culture was incubated for 5 h in the shaking incubator at 37 oC with shaking 200 rpm with 500 ul samples taken every hour, labelled 1, 2, 3, 4 and 5 h incubation and stored on ice. In the end 200 ul of each duplicate sample was aliquoted into black clear bottom 96 well plate. Duplicate samples of LB media containing both antibiotics were used as a negative control. The OD600 and fluorescence (ex485, em520) were recorded using PHERAstar FS (BMG Labtech) 96well plate reader. Recorded results were normalised to LB media and average results of two duplicate samples are presented in table 1 and table 2 and figure 5 and figure 6 respectively. As shown in our results our sRNA construct inhibit translation of the reporter GFP molecule in E.coli during the five hour incubation period used but we also noticed that as well as inhibiting GFP translation it also somehow inhibits bacterial grow which is evident from the OD600 data..

Table 1. OD600 of Xl1Blue E.coli cell culture harbouring reporter plasmid pSB4K5_BBa_K608010 and the sRNA expression plasmid pSB1C3_BBa_K2968005, as well as the pSB4K5_BBa_K608010 and the negative control plasmid pSB1C3_BBa_K2968003 respectively.

Table 2. GFP Fluorescence (ex485 nm, em520 nm) of Xl1Blue E.coli cell culture harbouring reporter plasmid pSB4K5_BBa_K608010 and the sRNA expression plasmid pSB1C3_BBa_K2968005, as well as the pSB4K5_BBa_K608010 and the negative control plasmid pSB1C3_BBa_K2968003 respectively.

Figure 5. OD600 of Xl1Blue E.coli cell culture harbouring reporter plasmid pSB4K5_BBa_K608010 and the sRNA expression plasmid pSB1C3_BBa_K2968005, as well as the pSB4K5_BBa_K608010 and the negative control plasmid pSB1C3_BBa_K2968003 respectively.

Figure 6. GFP Fluorescence (ex485 nm, em520 nm) of Xl1Blue E.coli cell culture harbouring reporter plasmid pSB4K5_BBa_K608010 and the sRNA expression plasmid pSB1C3_BBa_K2968005, as well as the pSB4K5_BBa_K608010 and the negative control plasmid pSB1C3_BBa_K2968003 respectively.