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.

Figure 1: BBa_K2968002 expressing sRNA secondary structure predicted using the RNAFold WebServer. 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 3.

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 5 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.

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

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 degrees C 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 degrees C 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.

Time (hr)	pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968000	pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968001
0	0.094	0.076
1	0.150	0.119
2	0.274	0.210
3	0.359	0.299
4	0.501	0.451
5	0.618	0.614

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.

Time (hr)	pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968000	pSB4K5_BBa_K608011 and pSB1C3_BBa_K2968001
0	65.5	12.5
1	146	25
2	384	45
3	526.5	66.5
4	780	103.5
5	1077.5	140.5

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.

BBa_K2968005 Validation

To generate functional sRNA molecule with the secondary structure shown in figure 8 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 9.

Figure 8: BBa_K2968005 expressing sRNA secondary structure predicted using RNAWebFold server. The mRNA target binding region is highlighted in pink and the sRNA scaffold in teal.

Figure 9: 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 10. The Spot42 scaffold is the 51 bp region of the E.coli sRNA.

Figure 10: 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 11 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 11: Spot42 sRNA colouring plate.

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

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 degrees C 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 degrees C 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 3 and table 4 and figure 13 and figure 14 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 3: 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.

Time (hr)	pSB4K5_BBa_K608010 and pSB1C3_BBa_K2868003	pSB4K5_BBa_K608010 and pSB1C3_BBa_K2868005
0	0.074	0.074
1	0.120	0.110
2	0.207	0.136
3	0.310	0.172
4	0.463	0.229
5	0.608	0.325

Table 4: 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.

Time (hr)	pSB4K5_BBa_K608010 and pSB1C3_BBa_K2868003	pSB4K5_BBa_K608010 and pSB1C3_BBa_K2868005
0	239	34
1	550	50
2	1149	92
3	2031	143
4	3134	241
5	4376	401

Figure 13: 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 14: 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.

BBa_K2968008 Validation

To generate functional sRNA molecule with the secondary structure shown in figure 15 our team has created this composite part consisting of three basic parts: BBa_J23100 promoter, BBa_K2968014 mRNA target binding region and BBa_K2968012 RprA sRNA scaffold as shown in figure 16.

Figure 15: BBa_K2968008 expressing sRNA secondary structure predicted using the RNAWebFold server. The mRNA target binding region is highlighted in pink and the sRNA scaffold in teal.

Figure 16: Details of the BBa_K2968008 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 17. The RprA scaffold is the 48 bp region of the E.coli sRNA.

Figure 17: RprA sRNA construct target binding region to the GFP mRNA.

To validate our BBa_K2968008 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 18 panel E. Visual analysis indicates that the sRNA expressed from the BBa_K2968008 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_K2968008 is functioning as initially expected by our team.

Figure 18: RprA sRNA colouring plate.

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

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 5 and table 6 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 slightly inhibits bacterial grow which is evident from the OD600 data.

Table 5: OD600 of Xl1Blue E.coli cell culture harbouring reporter plasmid pSB4K5_BBa_K608010 and the sRNA expression plasmid pSB1C3_BBa_K2968008, as well as the pSB4K5_BBa_K608010 and the negative control plasmid pSB1C3_BBa_K2968006 respectively.

Time (hr)	pSB4K5_BBa_K608010 and pSB1C3_BBa_K2868006	pSB4K5_BBa_K608010 and pSB1C3_BBa_K2868008
0	0.071	0.082
1	0.116	0.112
2	0.178	0.169
3	0.290	0.234
4	0.462	0.329
5	0.586	0.469

Table 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_K2968008, as well as the pSB4K5_BBa_K608010 and the negative control plasmid pSB1C3_BBa_K2968006 respectively.

Time (hr)	pSB4K5_BBa_K608010 and pSB1C3_BBa_K2868006	pSB4K5_BBa_K608010 and pSB1C3_BBa_K2868008
0	246	31
1	480	42
2	912	53
3	1494	76
4	2470	130
5	3567	220

Figure 19: OD600 of Xl1Blue E.coli cell culture harbouring reporter plasmid pSB4K5_BBa_K608010 and the sRNA expression plasmid pSB1C3_BBa_K2968008, as well as the pSB4K5_BBa_K608010 and the negative control plasmid pSB1C3_BBa_K2968006 respectively.

Figure 20: 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_K2968008, as well as the pSB4K5_BBa_K608010 and the negative control plasmid pSB1C3_BBa_K2968006 respectively.

Team:KCL UK/Composite Part

Composite Parts

GcvB sRNA BioBricks:

BBa_K2968000

BBa_K2968001

BBa_K2968002

Spot42 sRNA BioBricks:

BBa_K2968003

BBa_K2968004

BBa_K2968005

RprA sRNA BioBricks:

BBa_K2968006

BBa_K2968007

BBa_K2968008

Part Validation:

BBa_K2968001 Validation: