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Parts

Basic Parts

BBa_K1689013 — N-terminal of lactamase fused with dCas9

BBa_K1689014 — dCas9 fused with С-terminal segment of beta-lactamase

We examined expression of β-lactamase gene under control of the inducible lacI promoter (BBa_K1189007). We performed antibiotic assay and IPTG assay to look how gene expression is affected by different conditions. In our experiments we used two E.coli strains: DH5α and BL21.

Figure 1. Growth curve of DH5α strain before and after transformation with a plasmid with β-lactamase gene under control of the inducible lacI promoter (BBa_K1189007).

Figure 2. Growth curve of BL21 strain before and after transformation with a plasmid with β-lactamase gene under control of the inducible lacI promoter (BBa_K1189007).

Fig.1 and Fig.2 show difference in growth of E.coli culture with and without the plasmid carrying β-lactamase expression cassette. We expected that E.coli culture with the plasmid will have a lower growth rate compared to the growth rate of E.coli culture without the plasmid (due to stress caused to cells by hosting a plasmid). In opposite to case with DH5a strain, results for BL21 correlate with our theoretical assumption

Figure 3. Growth curve of DH5α strain in LB containing Amp [100 mkg/ml] and Cm [34 mkg/ml] before and after transformation with a plasmid with β-lactamase gene under control of the inducible lacI promoter (BBa_K1189007.

Figure 4. Growth curve of BL21 strain in LB containing Amp [100 mkg/ml] and Cm [34 mkg/ml] before and after transformation with a plasmid with β-lactamase gene under control of the inducible lacI promoter (BBa_K1189007).

Fig.3 and Fig.4 show that presence of Amp and Cm inhibits the growth of E.coli strains without plasmid carrying β-lactamase expression cassette . Strains with plasmid can grow in the presence of both Amp and Cm. Resistance to Amp is provided by β-lactamase and resistance to Cm is provided by Cm resistance gene in pSB1C3.

Figure 5. Growth curve of DH5α strain in LB containing different antibiotics (Amp [100 mkg/ml], Cm [34 mkg/ml], Kan [50 mkg/ml]) after transformation with a plasmid with β-lactamase gene under control of the inducible lacI promoter (BBa_K1189007).

Figure 6. Growth curve of BL21 strain in LB containing different antibiotics (Amp [100 mkg/ml], Cm [34 mkg/ml], Kan [50 mkg/ml]) after transformation with a plasmid with β-lactamase gene under control of the inducible lacI promoter (BBa_K1189007).

Fig.5 and Fig.6 show different growth rate for E.coli strains in LB containing different antibiotics. For both BL21 and DH5α strains maximum growth rate was reached in LB without any antibiotics. Minimum growth rate was demonstrated in the presence of Kan (because of absence of any resistance to Kan antibiotic). Growth rate of both strains was slightly lower in the presence of both Cm and Amp than in the presence of only Cm or Amp. Gene expression is observed without induction with IPTG due to promoter leakage.

Figure 7. Growth curve of DH5α strain in LB with Amp [100 mkg/ml] and Cm [34 mkg/ml] containing different IPTG concentrations (0 mM, 0.05 mM, 0,1 mM, 3 mM, 5 mM IPTG in LB) after transformation with a plasmid with β-lactamase gene under control of the inducible lacI promoter (BBa_K1189007).

Figure 8. Growth curve of BL21 strain in LB with Amp [100 mkg/ml] and Cm [34 mkg/ml] containing different IPTG concentrations (0 mM, 0.05 mM, 0,1 mM, 3 mM, 5 mM IPTG in LB) after transformation with a plasmid with β-lactamase gene under control of the inducible lacI promoter (BBa_K1189007).

Fig.7 and Fig.8 show different growth curve rate for E.coli cultures in LB containing different IPTG concentrations. As seen from these pictures, growth rate with and without IPTG induction did not differ. From this data, we can conclude that the lacI promoter can be used without induction and expression can be observed due to promoter leakage.

Improved Parts

BBa_K3028000 — dCas9 fused with N-terminal segment of beta-lactamase + BBa_B0015

BBa_K3028001 — dCas9 fused with С-terminal segment of beta-lactamase + BBa_B0015

BBa_K3028002 — Two BBa_K1689000 parts carrying sgRNA spacer + fragment with targets for different CRISPR/Cas pairs
This part is a composite part of two BBa_K1689000 parts, which are sgRNA generators with already integrated spacers for two CRISPR/Cas complexes from Streptococcus pyogenes. The part is composed together with a fragment carrying targets for each of sgRNA. Targets are located on 21bp from each other and have configuration of both PAM sites located out of targets.
This part is to be applied for screening of split-reporter systems activities associated with CRISPR/Cas complexes.

BBa_K3028003 — Two BBa_K1689000 parts carrying sgRNA spacer + fragment with targets for different CRISPR/Cas pairs
This part is a composite part of two BBa_K1689000 parts, which are sgRNA generators with already integrated spacers for two CRISPR/Cas complexes - one from Streptococcus pyogenes and other from Staphylococcus aureus. The part is composed together with a fragment carrying targets for each of sgRNA. Targets are located on 21bp from each other and have the configuration of both PAM sites located out of targets.
This part is to be applied for the screening of split-reporter systems activities associated with CRISPR/Cas complexes.