We have obtained pSB1C3 plasmids BBa_K608010 and BBa_K608011 from iGEM 2019 Kit. pSB1C3_BBa_K608010 contains a medium promoter from the constitutive promoter family combined with a 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.

Since the plasmids provided from the iGEM 2019 kit were not compatible with our sRNA, we used restriction digests to sub-clone the parts BBa_K608010 and BBa_K608011 into the plasmids, pSB4K5. Plasmid cloning was carried out in order to easily move the parts from pSB1C3 to 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, which has a copy of number of approximately 5 per cell. Based on such differences in origin of replication and the number of copy per cell, we hypothesised that psB1C3_BBa_K608010 and psB1C3_BBa_K608011 would have approximately 100 times stronger gene expression than pSB4K5_BBa_k608010 and pSB4K5_BBa_k608011.

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. This visual colour development of colonies on plate showed strong GFP expression in pSB4K5_BBa_K608010 strains (D), weak expression in pSB4K5_BBa_K608011(C). Additionally, stronger GFP expression was observed in BBa_K608010 strains (D) in comparison to pSB1C3_BBa_K608010 (B), suggesting that pSB4K5 is a higher copy number plasmid than originally thought. This has also been noted by Team Warwick in 2015 and Team Uppsala in 2012.

Figure 3: A plate with XL1-Blue E.coli transformed with plasmid pSB1C3_BBa_K608011 (A) and pSB1C3_BBa_K608010 (B) seen under UV light.

Figure 4: A plate with XL1-Blue E.coli transformed with plasmid pSB4K5_BBa_K608011 (C) and pSB4K5_BBa_K608010 (D) seen under UV light

We collected samples from liquid cultures of each plasmid with different parts, BBa_K608010 and BBa_K608011 at baseline and every hour for 5 hours (24 samples in all). Detailed methods of the experiment can be found in our Project Design and Notebook section. We measured the levels of optical density (OD) (shown in Table 1 and Graph 1) and GFP fluorescence (shown in Table 2 and Graph 2) using a plate reader in order to measure arbitrary unit fluorescence measurements into standard comparable units for functional analysis. The table below shows the measurements taken at Optical Density 600nm for BBa_K608010 and BBa_K608011.

Optical Density:

Table 1: Optical density (OD600nm) Measurements for BBa_K608010 and BBa_K608011 BioBricks with the two plasmids pSB4K5 and pSB1C3.

Optical Density (600nm)
Time	pSB1C3_BBa_K608011	pSB1C3_BBa_K608010	pSB4K5_BBa_K608011	pSB4K5_BBa_K608010
Oh	0.067	0.070	0.070	0.090
1h	0.122	0.108	0.145	0.118
2h	0.229	0.186	0.255	0.203
3h	0.328	0.278	0.437	0.335
4h	0.470	0.425	0.686	0.505
5h	0.610	0.591	0.672	0.630

Graph 1: Optical density (OD600nm) Measurements for BBa_K608010 and BBa_K608011 BioBricks with the two plasmids pSB4K5 and pSB1C3.

All samples were measured in duplicate in order to minimise any experimental error. The optical density (OD600) results show steady increase in cell growth over time in all four plasmids. pSB4K5_BBa_K608011, a plasmid with a medium promoter and a medium ribosomal binding site showed the fastest growing rate until the 4th hour. pSB1C3_BBa_K608010, a plasmid with a medium promoter and a strong ribosomal binding site showed the lowest growing rate. When two plasmids had the same construct but different plasmid backbones, the plasmids with pSB4K5 backbone grew higher number of cells compared to the plasmids with pSB1C3 backbone. The results indicate that pSB4K5 is not a low copy number plasmid.

Fluorescence:

Table 2: Fluorescence Measurements for BBa_K608010 and BBa_K608011 BioBricks with the two plasmids pSB4K5 and pSB1C3.

Fluorescence
Time	pSB1C3_BBa_K608011	pSB1C3_BBa_K608010	pSB4K5_BBa_K608011	pSB4K5_BBa_K608010
Oh	38	155.5	52.5	269
1h	334.5	1108	157.5	637
2h	1111.5	2512.5	425.5	1464
3h	1626.5	3593	738	2499.5
4h	2199.5	5305.5	1126.5	3888
5h	2892	7057.5	1289	5308

Graph 2: Fluorescence Measurements for BBa_K608010 and BBa_K608011 BioBricks with the two plasmids pSB4K5 and pSB1C3.

We measured gene expression using reporter fusions with a fluorescent protein, allowing high time resolution monitoring and quantifying. All four plasmids showed linear increase in gene expression over time, pSB1C3_BBa_K608010 showing the highest rate of increase whereas pSB4K5_BBa_K608011 showing the lowest rate of increase. It is worth noticing that two plasmids with the lowest rate of increase both contain the part, BBa_K608011, a part with a medium promoter and a medium ribosomal binding site. The data suggests that different plasmid backbones affect the strength of gene expression; pSB1C3 resulting the higher level of gene expression compared to pSB4K5. We have characterised pSB4K5 using different antibiotic resistance. The results from the measurement of GFP Fluorescence suggest that pSB4K5 does not have a low copy number of origin.

Conclusion

The results suggest that pSB4K5 has a significantly higher copy number than the expected number from the literature. This characterisation of existing part correlated with the results from iGEM Team Uppsala University 2012, where they demonstrated that pSB4C5 does not have a low copy origin. Our results from this characterisation could expand and be applied to the whole pSB4x5 series, meaning that the pSB4x5 plasmids are not likely to have low copy number backbones as identified in the registry.

Team:KCL UK/Contribution

Contribution

We conducted characterisation of BioBricks BBa_K608010 and BBa_K608011 in order to experimentally validate the parts and aid better understanding for users.

BBa_K608010 BioBrick