Team:KCL UK/Demonstrate
Demonstrate
Our BioBricks
GcVB sRNA BioBricks
We have obtained pSB1C3 plasmids BBa_K608010 and BBa_K608011 from iGEM 2019 Kit. pSB1C3_BBa_K608010 contains medium promoter from the constitutive promoter family combined with strong Ribosomal Binding Sites (PR4) and GFP. GFP in BBa_K608010 was tagged to the promoter Ribosomal Binding Sites RBS domain as a reporter molecule in order to quantify gene expression. pSB1C3_BBa_K608011 contains medium promoter from the constitutive promoter family combined with medium RBS (PR5) and GFP as a reporter molecule to quantify gene expression.
Then we synthesised our sRNA, GcvB into three different plasmids; BBa_K2968000, BBa_K2968001 and BBa_K2968002. BBa_K2968000 was our negative control with a terminator with no functional sRNA (Figure 1), BBa_K2968001 expressed sRNA against medium RBS and GFP (Figure 2), and BBa_K2968002 expressed sRNA against strong RBS and GFP (Figure 3).
Then we decided to perform double plasmid transformation in order to create biobricks which have compatible origin of replication with GcvB. We used restriction digests to subclone the parts BBa_K608010 and BBa_K608011 into the plasmids, pSB4K5. Plasmid pSB4K5 was bounded by restriction sites present in the same orientation as plasmid pSB1C3 but contained different origin of replication. pSB1C3 is a high copy number plasmid with the origin of replication pUC10-derived pBM1, which has a copy number of approximately 500 per cell. pSB4K5 is a low copy number of plasmid with the origin of replication pSC101.
In order to visually compare and contrast the gene expression of BBa_K608010 and BBa_K608011, we co-trasnfected XL1Blue E.coli with pSB1Cs plasmid containing appropriate sRNA and pSB4K5 plasmid containing GFP and plated on LB agar plates containing both chloramphenicol and kanamycin antibiotics (Figure 4). This visual colour development of colonies on plate showed strong GFP expression in plasmid pSB4K5_BBa_K608010 and psB1C3_BBa_K2968000 strain (D), weaker expression in pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968000(A). Such difference in GFP expression suggests the importance of RBS strength in gene regulation. In comparison to two negative control (A, D), rest of the colonies with functional GcvB showed significantly less GFP expression with different intensity. Different degree of intensity in GFP expression exhibiting in different RBS binding affinity suggests that sRNA against RBS inhibits GFP expression.
The cells transformed with these constructs were grown in LB medium at 37℃ for 5 hours. This experimental design would allow us to assess the influence of the binding sensitivity of the RBS to GcvB. We collected samples every 1 hour for 5 hours and obtained fluorescence and OD 600 data by measuring the samples on a black 96well plate.
A. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968000
B. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968001
C. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968001
D. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968000
E. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968002
F. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968002
GcvB sRNA Optical Density
Table 1: Optical density (OD) measurements for the GcvB BioBricks at 600nm.
Figure 5: Optical density (OD) measurements for the GcvB BioBricks at 600nm..
We performed all our experiments in duplicates and the analysis of the OD600, which is an indication of bacterial division and growth, shows that only pSB1C3_BBa_K2968002 expressing GcvB sRNA construct and GFP has some negative effect on the bacterial growth. This might be due to the fact that our sRNA might inhibit not only the GFP expression from the pSB4K5_BBa_K608011 construct but also other native E.coli genes as our target ribosome binding site is a strong native bacterial ribosome binding site (Elowitz and Leibler, 2000).
GcvB sRNA Fluorescence
Table 2: Fluorescence measurements for the GcvB BioBricks.
Figure 6: Fluorescence measurements for the GcvB BioBricks.
Analysis of the GFP fluorescence has shown that our sRNA constructs efficiently downregulate GFP translation and thus protein expression. To our surprise, we have also noticed that the target binding region spanning only 12 nucleotides is sufficient enough to allow our sRNA inhibit the expression of the GFP as our synthetic GcvB sRNA targeting BBa_B0034 ribosomal binding site was also effective to inhibit expression of the GFP with the BBa_B0032 ribosomal binding site as well as the sRNA against BBa_B0032 ribosomal binding site was effective against the BBa_B0034 ribosomal binding site. Both sRNAs in the target binding region have identical 12 nucleotide sequence complementary to the GFP start codon as shown in the red box figure 1 and 2, and thus in our opinion the strength of the interaction between the sRNA and GFP mRNA in this region is enough to prevent ribosome binging. This should be considered when designing new synthetic sRNA.
Spot42 sRNA BioBricks
We have obtained pSB1C3 plasmids BBa_K608010 and BBa_K608011 from iGEM 2019 Kit. pSB1C3_BBa_K608010 contains medium promoter from the constitutive promoter family combined with strong Ribosomal Binding Sites (RBS) (PR4) and GFP. GFP in BBa_K608010 was tagged to the promoter RBS domain as a reporter molecule in order to quantify gene expression. pSB1C3_BBa_K608011 contains medium promoter from the constitutive promoter family combined with medium RBS (PR5) and GFP as a reporter molecule to quantify gene expression.
We synthesised our sRNA, Spot 42 into three different plasmids; BBa_K2968003, BBa_K2968004 and BBa_K2968005. BBa_K2968003 was our negative control with a bacterial terminator with no sRNA (Figure 9), BBa_K2968004 expressed sRNA against medium RBS and GFP (Figure 10), and BBa_K2968005 expressed sRNA against strong RBS and GFP (Figure 11).
Then we decided to perform double plasmid transformation in order to create biobricks which have compatible origin of replication with Spot 42. We used restriction digests to subclone the parts BBa_K608010 and BBa_K608011 into the plasmids, pSB4K5. Plasmid pSB4K5 was bounded by restriction sites present in the same orientation as plasmid pSB1C3 but contained different origin of replication. pSB1C3 is a high copy number plasmid with the origin of replication pUC10-derived pBM1, which has a copy number of approximately 500 per cell. pSB4K5 is a low copy number of plasmid with the origin of replication pSC101.
In order to visually compare and contrast the gene expression of BBa_K608010 and BBa_K608011, we co-trasnfected XL1Blue E.coli with pSB1Cs plasmid containing appropriate sRNA and pSB4K5 plasmid containing GFP and plated on LB agar plates containing both chloramphenicol and kanamycin antibiotics (Figure 12). This visual colour development of colonies on plate showed the strongest GFP expression in plasmid pSB4K5_BBa_K608010 and psB1C3_BBa_K2968003 strain (D) which contains medium promoter, strong RBS and GFP. The importance of the RBS strength can be visualised when comparing the GFP expression of two identical constructs with different RBS; between A (medium RBS) and D (strong RBS). The colonies grown from the constructs with functional Spot 42 showed significant weaker expressions compared to the two negative control A and D. The intensity of GFP expression varied between the four colonies (B, C, E, F) depending on the specificity of the constructs, defining the degree of inhibition. Our results suggest that Spot 42 is down regulation gene expression.
A. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968003
B. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968004
C. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968004
D. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968003
E. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968005
F. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968005
Spot42 Optical Density:
Table 3: Optical density (OD) measurements for the Spot42 BioBricks at 600nm.
Figure 13: Optical density (OD) measurements for the Spot42 BioBricks at 600nm.
Our results show that Spot42 scaffold with our synthetic target binding region also inhibits bacterial growth. We have also noticed that this inhibition is sequence dependent as different combinations of the sRNA scaffold and the target binding region had different degree of the bacterial growth inhibition. This is quite surprising result for us which requires further investigation.
Spot42 Flouresence:
Table 4: Fluorescence measurements for the Spot42 BioBricks.
Figure 14: Fluorescence measurements for the Spot42 BioBricks.
The results of the GFP fluorescent as an indicator of the degree to which our synthetic Spot42 sRNA constructs inhibiting mRNA translation have shown that some constructs did not demonstrate complete inhibition of the mRNA translation as for some constructs we observed significant level of the GFP fluorescence. This observation in combination with the OD600 data might indicate that some of our Spot42 constructs might have additional targets in the E.coli and thus there is not enough sRNA expression to completely shut down the production of the GFP. Further investigations of alternative Spot42 constructs might show the reason for our observations.
RprA sRNA BioBricks
We have obtained pSB1C3 plasmids BBa_K608010 and BBa_K608011 from iGEM 2019 Kit. pSB1C3_BBa_K608010 contains medium promoter from the constitutive promoter family combined with strong Ribosomal Binding Sites (RBS) (PR4) and GFP. GFP in BBa_K608010 was tagged to the promoter RBS domain as a reporter molecule in order to quantify gene expression. pSB1C3_BBa_K608011 contains medium promoter from the constitutive promoter family combined with medium RBS (PR5) and GFP as a reporter molecule to quantify gene expression.
We synthesised our sRNA, RprA into three different plasmids; BBa_K2968006, BBa_K2968007 and BBa_K2968008. BBa_K2968006 was our negative control with a bacterial terminator with no sRNA expressing (Figure 15), BBa_K2968004 expressed sRNA against medium RBS and GFP (Figure 16), and BBa_K2968005 expressed sRNA against strong RBS and GFP (Figure 17).
Then we performed double plasmid transformation in order to create biobricks which have compatible origin of replication with RprA. We used restriction digests to subclone the parts BBa_K608010 and BBa_K608011 into the plasmids, pSB4K5. Plasmid pSB4K5 was bounded by restriction sites present in the same orientation as plasmid pSB1C3 but contained different origin of replication. pSB1C3 is a high copy number plasmid with the origin of replication pUC10-derived pBM1, which has a copy number of approximately 500 per cell. pSB4K5 is a low copy number of plasmid with the origin of replication pSC101.
In order to visually compare and contrast the gene expression of BBa_K608010 and BBa_K608011, we co-trasnfected XL1Blue E.coli with pSB1Cs plasmid containing appropriate sRNA and pSB4K5 plasmid containing GFP and plated on LB agar plates containing both chloramphenicol and kanamycin antibiotics (Figure 18). This visual colour development of colonies on plate showed the strongest GFP expression in plasmid pSB4K5_BBa_K608010 and psB1C3_BBa_K2968003 strain (D) which contains medium promoter, strong RBS and GFP. The importance of the RBS strength can be visualised when comparing the GFP expression of two identical constructs with different RBS; between A (medium RBS) and D (strong RBS). The colonies grown from the constructs with RprA showed significant weaker expressions compared to the two negative control A and D.
A. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968006
B. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968007
C. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968007
D. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968006
E. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608010 and pSB1C3_BBa_K2968008
F. XL1-Blue E.coli transformed with plasmids pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968008
RprA sRNA Optical Density:
Table 5: Optical density (OD) measurements for the RprA BioBricks at 600nm.
Figure 19: Optical density (OD) measurements for the RprA BioBricks at 600nm.
The OD600 measurements taken every hour for 6 hours show a clear relationship between sRNA inhibition of RBS and bacterial growth. Although the results do not exhibit the same degree of inhibition as shown in Spot 42 BioBricks, the rate of bacterial growth in the constructs with RprA against medium RBS and strong RBS both show significant decrease in cell growth compared to the construct with a terminator and no functional RprA.
RprA Flouresence:
Table 6: Fluorescence measurements for the RprA BioBricks.
Figure 20: Fluorescence measurements for the RprA BioBricks.
Fluorescence analysis of the GFP expression have shown that our RprA sRNA construct efficiently inhibit expression of the GFP in E.coli. Although some inhibition of the bacterial growth was observed, in our opinion the RprA sRNA constructs are effective and could be used to constructs novel sRNA based therapeutic molecules or use them for metabolic engineering of E.coli and other future applications.
