Part:BBa_K2968001
sRNA GcvB targeting BBa_B0032 and BBa_E0040 start codon
This sRNA has promoter target binding region for BBa_B0032 and BBa_E0040 start codon
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
KCL iGEM 2019
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_K2968013 mRNA target binding region and BBa_K2968010 GcvB sRNA scaffold as shown in figure 2. Part BBa_K2968010 codes for GcvB sRNA scaffold that is based on the native E.coli ncRNA GcvB. The nucleotide sequence obtained from the BioCyc database: https://biocyc.org/gene?orgid=ECOLI&id=GCVB-RNA
Figure 1. BBa_K2968002 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_K2968001 sRNA construct design
To create this part and introduce the sRNA target binding region, we used two primers and QuikChange II XL Site-Directed Mutagenesis Kit as recommended by the manufacturer Agilent.
As a template we used BBa_K2968000 part cloned into pSB1C3 plasmid that we created to use as a negative control for our experiments as it has a transcription terminator instead of a target binding region. DNA sequence of the BBa_K2968000 part with primers complementarity regions is shown in figure 2.
Figure 3. DNA sequence of the BBa_K2968000. BBa_J23100 promoter is highlighted in turquoise and the GcvB sRNA scaffold sequence in yellow. Regions complementary to the primers GcvBFror and SDMsRNARev are in underlined and shown in bold.
The BBa_J23100 promoter is a strong promoter from the constitutive promoter family designed by John Anderson, iGEM2006_Berkeley. BBa_K2968013 part is a complementary part to the BBa_K608011 that has BBa_B0032 Ribosome Binding Site (RBS) and BBa_E0040 GFP gene part as shown in figure 4. The GcvB scaffold is the 84 bp region of the E.coli sRNA.
Figure 4. GcvB sRNA construct target binding region to the GFP mRNA.
To validate our BBa_K2968001 composite part, we transformed Xl1Blue E.coli cells with the pSB1C3 plasmid harbouring this part together with the reporter GFP plasmid pSB4K5 containing BBa_K608011 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_K2968001 part inhibits GFP reporter molecule expression Figure 5 panel B as compare to the pane A where E.coli was co-transformed with the GFP reporter plasmid and non sRNA expression plasmid. This result confirms that our part BBa_K2968001 is functioning as initially expected by our team.
Figure 5. Analysis of the function of the GcvB 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_K2968000; Panel B. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968001; Panel C. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968001; Panel D. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968000; Panel E. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968002; Panel F. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968002
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 6 and figure 7 respectively. As shown in our results our sRNA construct completely inhibit translation of the reporter GFP molecule in E.coli during the five hour incubation period used.
Table 1. OD600 of Xl1Blue E.coli cell culture harbouring reporter plasmid pSB4K5_BBa_K608010 and the sRNA expression plasmid pSB1C3_BBa_K2968001, as well as the pSB4K5_BBa_K608011 and the negative control plasmid pSB1C3_BBa_K2968000 respectively.
Table 2. GFP Fluorescence (ex485 nm, em520 nm) of Xl1Blue E.coli cell culture harbouring reporter plasmid pSB4K5_BBa_K608011 and the sRNA expression plasmid pSB1C3_BBa_K2968001, as well as the pSB4K5_BBa_K608011 and the negative control plasmid pSB1C3_BBa_K2968000 respectively.
Figure 6. OD600 of Xl1Blue E.coli cell culture harbouring reporter plasmid pSB4K5_BBa_K608011 and the sRNA expression plasmid pSB1C3_BBa_K2968001, as well as the pSB4K5_BBa_K608011 and the negative control plasmid pSB1C3_BBa_K2968000 respectively.
Figure 7. GFP Fluorescence (ex485 nm, em520 nm) of Xl1Blue E.coli cell culture harbouring reporter plasmid pSB4K5_BBa_K608011 and the sRNA expression plasmid pSB1C3_BBa_K2968001, as well as the pSB4K5_BBa_K608011 and the negative control plasmid pSB1C3_BBa_K2968000 respectively.
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