Characterization of BBa_E0020 (ECFP):
The BioBrick BBa_E0020 was characterized by using the BioBrick BBa_I13600 as a generator of the ECFP(Engineered Cyan Fluoroscent Protein) and due to availiability in the 20 D well of the Distribution Kit Plate 2. Further, this part was incorporated in BL21 DE3 for the better expression and folding of the protein and was subjected for protein extraction.
Fig.1,SDS PAGE Gel for ECFP Characterization. As can be seen from the marked band in the SDS PAGE lies in between the 25 KDa and 32 KDa. The calculated size of the protein of interest is around 28 KDa which is very close to the theoretical one i.e. 26.9 KDa.
Fig.2
SDS PAGE after Ammonium sulfate Purification Assay. As can be seen from, there are intensively bright bands at the expected size i.e. 27 KDa in 55% and 60% (w/V) saturation by ammonium sulfate.
Artificially derived from GFP, the engineered cyan fluorescent protein (ECFP) is commonly used as a fluorescent marker in molecular biology studies due to its photostability, high lifespan and relatively low maturation times. To characterize the fluorophore we first performed a full wavelength scan to obtain the excitation and emission spectra of the protein.
Fig (1) : Excitation and Emission Spectra for ECFP
CcaSR System:
This system consists of two subunits: CcaS-GP2 (BBa_K3165039) and CcaR-Ho1-PcyA (BBa_K3165036).The CcaR-Ho1-PcyA subunit was digested with EcoRI and PstI. Similarly, the plasmid pSB1C3 was digested with the same enzymes. Now, these two parts were ligated together and the ligated DNA was used for the transformation of DH5α strain of Escherichia coli. Colonies were randomly chosen from these transformed plates with chloramphenicol resistance. The figures mentioned below show the enumerated colonies on which plasmid miniprep was performed. These extracted plasmids were then digested with the same restriction endonucleases in order to check for the correct size of the inserted fragment.
Further, from the images, it can be concluded that colony numbers 6, 7, 8, 9 and 18 had inserted fragments of the correct size. We cross-checked the results by doing the colony PCR on only those colonies as the quality of digest wasn't very good.
CcaR-Ho1-PcyA screening colonies by digestion from Colony 1 to Colony 9
CcaR-Ho1-PcyA screening colonies by digestion from Colony 1' to Colony 8'
CcaR-Ho1-PcyA screening colonies by digestion from Colony 1'' to Colony 6''
CcaR-Ho1-PcyA screening colonies by digestion from Colony 7'' to Colony 10'' and Colony 18''
CcaR-Ho1-PcyA screening colonies by digestion of Colony 9'', Colony 10'' and Colony 18''
OPTO-T7 System:
This system consists of two subunits: T7-NMAG (BBa_K3165050) and PMAG-Gp2 (BBa_K3165052). We PCR amplified the PMAG-GP2 fragment (as shown in the figure below):
After the PCRing of the PMAG-GP2, it is digested with EcoRI and PstI. Similarly, the plasmid pBS1C was digested with the same enzymes. Now, these two parts were ligated together and the ligated DNA was used for the transformation of DH5α strain of Escherichia coli.
Colonies were randomly chosen from these transformed plates with ampicillin and spectinomycin resistance.
Colonies were chosen from these plates and plasmid miniprep was performed. The extracted plasmid was digested with EcoRI restriction enzyme to check for the right size of the linearized ligated plasmid.
Ligated pBS1C and pBS4S plasmid digested with EcoRI
The plasmids pBS1C and pBS4S digested to obtain linearized backbones
CcdAB Antitoxin-Toxin System
Expression and Characterisation
This part was incorporated in Top10 G (gryA) R462C which is one of the strains that can be used to produce CcdB L83S because of the mutation in Gyrase A making the cells tolerant to it.
The secondary culture was induced with L-Arabinose and a total cell lysate was made by following the protein extraction protocol. Further, an uninduced sample was also separated as a control to check leaky transcription.
The supernatant is subjected to protein purification by incubation in the CcdA column and later eluted to get 10 different samples that are loaded in the SDS PAGE mentioned.
Fig.1 - SDS PAGE Gel 1 showing the elutes for the purified CcdB L83S mutant protein.
Fig.2 - SDS PAGE Gel 2 showing the elutes for the purified CcdB L83S mutant protein.
It can be seen from the SDS PAGE image that there is a very faint band in the well containing the uninduced sample. So, we concluded a very low level of leaky transcription which further can be reduced by the addition of glucose to the culture as glucose acts as a repressor.
Also, the faint band in the pellet section can be used to evaluate the quality of the protein extraction.
As quite evident from the gel, the size of the protein is 11.7 kDa which matches the theoretical value.
Determination of the melting point of the CcdB L83S Protein
We also determined the melting point of the CcdB L83S mutant by performing Thermal Shift Assay.
Fig.3 The full Thermal Shift Assay graph for determining the melting point of CcdB L83S mutant protein.
Fig.4 The zoomed in picture of the plot as mention in "Fig.3" Thermal Assay graph for determining the melting point of CcdB L83S mutant protein.
As can be seen from Fig.3 and Fig.4, the melting point of the CcdB L83S mutant protein is calculated to be 43.03oC. When compared to the melting point of the wild type CcdB protein i.e. 63.8oC, the CcdB L83S protein has a much lower melting temperature. Hence, we concluded that the core mutation resulted in the instability of the CcdB L83S mutant.
In addition to this, Growth Assay can be used on strains like Top10 PJAT to draw the same conclusion i.e. the mutant CcdB L83S is less stable than it's wild type. So, this mutant can be used in regulating the bacterial population to an extent.
Growth Assay of CcdB L83S Protein mutant
To show that CcdB L83S is not as potent as the wild type protein, Top 10 pJET strain of E. coli containing the CcdB L83S gene was grown on LB agar plates with gentamicin and ampicillin resistance and containing various concentrations of arabinose and glucose.
Fig.5 - The plate shows the colonies of pJAT strain grown with no amount of arabinose or glucose.
We can see in Fig.5 that when LB agar plate with no inducer or repressor was used, colonies of bacteria were obtained, which validated the hypothesis that the CcdB L83S mutant can be grown in strains of E. coli which have no GyraseA subunit mutation.
Fig.6 - The plates shown have a gradually decreasing concentration of glucose towards the left. A trend can be seen that the colony size increases from left to right.
As can be seen from Fig.6, in the presence of glucose, the bacteria grew with at its normal rate and no effects were seen. It can further be noticed that the spots were more prominent at higher concentrations of glucose, which is valid as glucose is a repressor and it reduces the leaky transcription of the CcdB L83S protein.
Fig.7 - The plates shown have a gradually increasing concentration of arabinose towards the right. A trend can be seen that the colony size idecreases from left to right.
From Fig.7, we can see that despite the presence of arabinose in plates, we were able to visualize very small-sized colonies. But, at higher concentrations of arabinose, these colonies weren't able to grow.
This shows that CcdB L83S can function as a bacteriostatic protein or a bactericidal toxin under the right conditions, and can be used in strains of E. coli not containing mutations in Gyrase A subunit.
