Difference between revisions of "Part:BBa K1189007"

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−	=<b><i>In vivo</i> protein expression assay</b>=	+	===<b><i>In vivo</i> protein expression assay</b>===
	Our first goals for our improvement experiments, was to demonstrate that our improved part can be functional in vivo, after activation from our trigger sequence, and also prove that it is able to reach the expression levels of the initial part (BBa_K1189007).		Our first goals for our improvement experiments, was to demonstrate that our improved part can be functional in vivo, after activation from our trigger sequence, and also prove that it is able to reach the expression levels of the initial part (BBa_K1189007).

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Revision as of 15:26, 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

Aim

Our project design, utilizes the ability of the β-lactamase enzyme to hydrolyze the chromogenic substrate, nitrocefin, resulting in a color change from yellow to red. Based on this ability of β-lactamase, we decided to improve an already existing Biobrick (BBa_K1189007) which corresponds to the β-lactamase gene regulated by an inducible lacI promoter. This Biobrick part is only functional in vivo, and cannot be expressed with any of the usual in vitro transcription translation kits, because of the lack of an appropriate promoter (recognized by either T3, T7, or SP6 polymerase). Aiming to fit this part into our cell-free system and make it functional in vitro, we firstly replaced the lacI, inducible by IPTG, promoter with a T7 constitutive promoter (BBa_J64997). In order to regulate its expression, just as the lacI promoter does, we incorporated a Toehold switch sequence upstream of the CDS of β-lactamase. To be able to prove our improvement upon this part, we used the chromogenic substrate nitrocefin, which changes color from yellow (380nm) to red (490nm) when hydrolyzed by the β-lactamase enzyme. We then gathered quantitative results for the hydrolysis of our substrate, Nitrocefin, by both parts, though frequent plate reader assays after both in vivo and in vitro expression of the above mentioned constructs.

Constructs' creation

For our experiments we created the composite part BBa_K2973007 that consists of a T7 promoter, Pardee’s Toehold Switch 32B [1], a β-lactamase gene and a T7 terminator. Furthermore, we designed the composite part BBa_K2973023 that consists of a T7 promoter, Pardee’s Trigger 32B, and a T7 terminator. These parts, including the prefix & suffix sequences, were ordered from IDT and cloned into their respective plasmids (pSB1K3 vector for the 32B Trigger and pSB1C3 for the lacI β-lactamase and the 32B Toehold β-lactamase), with restriction digestion & ligation.

In vivo protein expression assay

Our first goals for our improvement experiments, was to demonstrate that our improved part can be functional in vivo, after activation from our trigger sequence, and also prove that it is able to reach the expression levels of the initial part (BBa_K1189007).

Method

In order to produce measurable and reproducible data, we used 2 biological and 2 technical replicates for each construct. Aiming to test the binding efficiency between the Toehold switch regulating the β-lactamase enzyme and the Trigger32B, we co-transformed the two plasmids that contained these constructs into BL21 (DE3) cells. Moreover, BL21 (DE3) cells were transformed with the plasmid that contains the lacI-regulated β-lactamase construct, in order to compare our improvement hypothesis.

To ensure that the absorbance measured corresponds only to the enzymatic activity of β-lactamase, we included 5 controls in our experiments:

• LB medium only (no cells) and nitrocefin

• Empty BL21 (DE3) cells (no plasmid) and nitrocefin

• BL21 (DE3) cells containing the empty vector and nitrocefin

• BL21 (DE3) cells with Toehold32B- β-lactamase and nitrocefin

• BL21 (DE3) cells with lacI- β-actamase (BBa_K1189007) and nitrocefin

The workflow of our in vivo experiments was performed as described below:

1. We grew the cultures overnight in 5ml LB (~16h) at a shaking incubator, 37oC / 210rpm

2. The following morning, we measured the OD600 of the overnight cultures

3. We diluted all cultures to OD600 = 0.1 in LB medium

4. We then grew the cells at 37 oC /210 RPM until OD600=0.5 (~2h)

5. We diluted all cells to the same OD600 (e.g. 0.5)

6. We loaded 200ul of culture in a 96-well plate (2 technical replication each) and 40ul of the nitrocefin substrate (0,5 mM), in order to perform the enzymatic assay.

7. We finally measured the absorbance at 490nm (for nitrocefin hydrolysis) and 600nm (for cell growth) in a microplate reader. We shook between measurements.

The absorbance measurements were conducted every 2 min for 75 minutes at 490nm for the hydrolyzed nitrocefin substrate, and at 600nm for the cell growth.

Results

Figure 1. Expression of β-lactamase reporter gene in vivo. Error bars represent the standard deviation for n = 2 biological replications. The substrate (nitrocefin) hydrolysis (490nm) is divided by cell growth (600nm), in order to normalize all values.

Figure 2. β-Lactamase expression levels for t=73minutes. Error bars represent the standard deviation for n = 2 biological replications.The substrate (nitrocefin) hydrolysis (490nm) is divided by cell growth (600nm), in order to normalize all values.

Figure 3. Change of the cultures’ color from yellow to red due to the hydrolyzation of nitrocefin (in vivo)

In vitro protein expression assay

For the second part of our improvement experiments we wanted to demonstrate that our improved part is not only functional in vivo but also in cell-free systems. Furthermore, we proved that the initial part (LacI-regulated β-lactamase) is not functional in vitro and cannot be expressed with our in vitro translation kit (PURExpress In vitro Protein Synthesis Kit), as it cannot be expressed with any of the usual in vitro transcription translation kits due to the lack of an appropriate promoter (recognized by either T3, T7, or SP6 polymerase).

Method

In order to produce measurable and reproducible data, we used 2 technical replicates for each construct. Τhe constructs that were used during the experiment and their respective quantities are listed below:

• T7 β-lactamase (positive control) 75nM

• Toehold 32B β-Lactamase (negative control) 75nM

• Toehold 32B β-Lactamase 70ng + Trigger 32B 75nM

• Toehold 32B β-Lactamase 70ng + Trigger 32B 7nM

• LacI β-Lactamase (initial part) 75nM

Our in vitro experiments were performed with the PURExpress Ιn Vitro Protein Synthesis Kit provided by New England Biolabs (NEB). We followed the standard protocol for a typical 7ul PURExpress reaction. Each PURExpress reaction was incubated for 3 hours for each construct. Finally, we measured the absorbance levels of our samples every 30 seconds for 45minutes total, at 490nm to be able to have quantitive results for the hydrolyzation of our substrate (Nitrocefin).

Results

Figure 4. Expression of β-lactamase reporter gene in vitro. Error bars represent the standard deviation for n = 2 technical replications.

Figure 5. Colour-change from yellow to red due to the hydrolyzation of nitrocefin (in vitro).

Sequence and Features

Sequence and Features

Sequence and Features