Difference between revisions of "Part:BBa K1189007"

Revision as of 09:33, 17 October 2019 (view source) Juliakacher (Talk | contribs) ← Older edit		Revision as of 13:20, 19 October 2019 (view source) Tkontogiannis (Talk | contribs) Newer edit →
Line 342:		Line 342:
	</html>		</html>
		+
		+	==Improvement by iGEM Thessaly 2019==
−	<!-- Add more about the biology of this part here
−	===Usage and Biology===
	<!-- -->		<!-- -->

---

Revision as of 13:20, 19 October 2019

Beta-lactamase with His Tag under the control of the inducible lacI promoter

This part was built to allow for the extraction of Beta-lactamase with the his-tags added onto the BioBrick. The part was built with the lacI IPTG inducible promoter J04500, with RBS.

Applications of BBa_K1189007

In addition to that, we have purified our beta-lactamase ( BBa_K1189007 ) and our mobile TALE A linked to beta-lactamase construct ( BBa_K1189031 ) (Figure 2) and we have demonstrated that beta-lactamase retained its enzymatic activity for both proteins. We repeated a variation of ampicillin survival assay where we pretreated LB containing ampicillin and chloramphenicol with our purified TALE A linked to beta-lactamase ( BBa_K1189031 ). We then cultured bacteria in the treated LB that only carry resistance to chloramphenicol. Therefore, the bacteria are only able to survive if the our isolated protein retained its enzymatic abilities. We can show that the bacteria susceptible to ampicillin was able to grow in the presence of our purified construct protein ( BBa_K1189031 ), which means that we are expressing and purifying functional protein which is degrading the ampicillin (Figures 1 and 3). Figure 3 shows the OD at 24 hour time point from culturing where Figure 1 shows OD change over time. Both graphs show an increase in OD for cultures pre-treated with our protein demonstrating our protein is functional.

After verifying that TALE A -linker-beta-lactamase ( BBa_K1189031 ) retained enzymatic activity and was able to degrade ampicillin, we performed a colourimetric assay using benzylpenicillin as our substrate. We were able to see a colour change from red to yellow. This is because there is phenol red, a pH indicator, added to the substrate solution. Beta-lactamase hydrolyzes benzylpenicillin to penicillinoic acid, which changes the pH of the solution from alkaline to acidic. This pH change causes the phenol red to change from red to yellow. Our negative controls, to which benzylpenicillin was not added, remained red. We can also see the colour change correlate to the amount of purified TALE A linked to beta-lactamase present in each sample (Figure 5).

Beta-lactamase with His Tag under the control of the inducible lacI promoter

This part was built to allow for the extraction of Beta-lactamase with the his-tags added onto the BioBrick. The part was built with the lacI IPTG inducible promoter J04500, with RBS.

Applications of BBa_K1189007

In addition to that, we have purified our beta-lactamase ( BBa_K1189007 ) and our mobile TALE A linked to beta-lactamase construct ( BBa_K1189031 ) (Figure 2) and we have demonstrated that beta-lactamase retained its enzymatic activity for both proteins. We repeated a variation of ampicillin survival assay where we pretreated LB containing ampicillin and chloramphenicol with our purified TALE A linked to beta-lactamase ( BBa_K1189031 ). We then cultured bacteria in the treated LB that only carry resistance to chloramphenicol. Therefore, the bacteria are only able to survive if the our isolated protein retained its enzymatic abilities. We can show that the bacteria susceptible to ampicillin was able to grow in the presence of our purified construct protein ( BBa_K1189031 ), which means that we are expressing and purifying functional protein which is degrading the ampicillin (Figures 1 and 3). Figure 3 shows the OD at 24 hour time point from culturing where Figure 1 shows OD change over time. Both graphs show an increase in OD for cultures pre-treated with our protein demonstrating our protein is functional.

After verifying that TALE A -linker-beta-lactamase ( BBa_K1189031 ) retained enzymatic activity and was able to degrade ampicillin, we performed a colourimetric assay using benzylpenicillin as our substrate. We were able to see a colour change from red to yellow. This is because there is phenol red, a pH indicator, added to the substrate solution. Beta-lactamase hydrolyzes benzylpenicillin to penicillinoic acid, which changes the pH of the solution from alkaline to acidic. This pH change causes the phenol red to change from red to yellow. Our negative controls, to which benzylpenicillin was not added, remained red. We can also see the colour change correlate to the amount of purified TALE A linked to beta-lactamase present in each sample (Figure 5).

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.

Fig.6 and Fig.7 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

Fig.8 and Fig.9 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.

Fig.10 and Fig.11 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.

Fig.12 and Fig.13 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.

Improvement by iGEM Thessaly 2019

Sequence and Features

Sequence and Features

Sequence and Features