Overview
During our work we studied, followed and documented dozens of protocols widely known in the field or more specific and adapted to our own needs. Here we present all of them that we utilized and that guided us in our (sought-after) lab work concerning our 3 basic modules : the engineered peptide’s expression, the hydrogel’s preparation and lastly, the creation, recombination, and characterisation of the bacterial consortium and its products .
Peptide’s Experiments
Our desired engineered sequence was purchased by Genscript biotech Corporation, New Jersey and shipped to us cloned into a pEX-A128 plasmid vector using Xho1/Nde1 restriction enzymes. The below procedures were executed :
Transfer of the desired sequence into pET-29c plasmid vector
Introduction
This procedure aims to transform E.Coli bacteria cells so that we can amplify our purchased plasmid pEX-A128. TOP10 bacterial cells are known as the most popular E.Coli strain engineered for high transformation efficiency. They are suitable for cloning supercoiled DNA and allow stable replication of high-copy number plasmids.
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This procedure aims to isolate the now amplified purchased plasmids pEX-A128
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This procedure aims to digest both of the plasmids so that we can isolate and transfer our insert in between them. This method utilizes the restriction enzymes Nde1 and Xho1.
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This procedure aims to extract and isolate our desired insert from the Agarose Gel. Here, this method acts as an intermediate step into cloning the desired nt sequence from the purchased engineered plasmid (pEX-A128) to the target plasmid (pET-29c).
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This procedure aims to recombine the plasmid vector pET-29c with our desired insert. This technique is commonly used to ligate the ends of linear DNA molecules or combine different linear DNA molecules. The reaction is catalyzed by DNA ligase, with T4 DNA ligase used in this case. This protocol specifically details the ligation of DNA segments, such as PCR products or digested fragments, into the plasmid vector pET-29c(+).
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This procedure aims to transform E.Coli Bacteria Cells with our desired recombined vector to be able to amplify and store it. The same procedure described above is followed.
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This procedure aims to isolate the now amplified recombinant plasmid pET-29c
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This procedure is diagnostic and it is used so that we can be sure of the efficient recombination of pET-29c. This method utilizes the restriction enzymes Nde1 and Xho1.
Transformation of E.Coli BL21
Introduction
This procedure aims to transform E.Coli Bacteria Cells with our desired recombined vector to be able to express our desired insert. The BL21(DE3) strain is widely used for recombinant protein production due to its engineered capacity to produce T7 polymerase and its deficiency in Lon and OmpT proteases.
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This procedure aims to induce our insert’s expression with the use of IPTG. IPTG is a molecular analog of allolactose, which removes a repressor from the lac operon to induce gene expression. Here, bacteria of E. coli strain BL21(DE3), previously transformed with the recombinant plasmids pET-29c (+), are used to induce our peptides expression
Isolation and Purification of produced peptide
Introduction
This procedure aims to isolate our produced peptide due to its interaction of its (6xHis)-COOH Tag with a Ni-Column. Affinity chromatography is based on the specific and reversible binding of proteins to ligand molecules attached to an inactive chromatography material.
Identification of produced product - peptide
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We followed this procedure in order to quantify the expressed peptide’s concentration in samples so we could prepare the SDS-PAGE samples to be run in the next procedure.
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This procedure aims to identify the expression of our desired insert (peptide) with the qualitative method of SDS-PAGE. This technique allows the separation of proteins based on their molecular weight.
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This procedure aims to stain the bands of the peptide created from the SDS-Page procedure. This method is based on the interaction of Coomassie Brilliant Blue R-250 dye with proteins, leading to the formation of a coloured complex of cyan colour
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TEV Protease is recommended for cleavage of a His-tag following purification by Affinity Chromatography (Ni Spin Columns). It is noted that while designing our peptide sequence we must have incorporated the amino acid sequence recognized by the protease enzyme appropriately
Hydrogel’s Experiments
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This procedure is used for the polymerization of chitosan, pectin and chitosan and pectin respectively under acidic conditions so that our control hydrogels can be formed.
Microorganisms’ Experiments
Amplification of plasmid vector
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This procedure aims to transform E.Coli bacteria cells so that we can amplify plasmid vector. TOP10 and even DH5a bacterial cells are known E.Coli strains with high transformation efficiency. They are suitable for cloning supercoiled DNA and allow stable replication of high-copy number plasmids.
Introduction
This technique is used for the extraction of plasmids I8, I8’ C12, C12’, E12, E12’, K8, K8’, M8, M8’, E9, E9’, G8, G8’, A12, A12’ of the iGEM Kit, from host E. coli cells. Below the Monarch® Plasmid Miniprep Kit is followed so that we can isolate our plasmid vector that has been amplified.
Construction recombinant plasmid vectors, amplification and isolation : Our part’s DNA sequences were designed and purchased from Twist Bioscience, California. We then inserted them into a plasmid vector () , cloned them into E.Coli Systems and isolated them.
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This procedure is carried out to determine and identify the results of the plasmid’s recombination. We used kb markers and tried to match the bands that occurred after this method’s execution to our expected nt size.
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Agarose Gel Electrophoresis is carried out as a diagnostic procedure for confirmation of the successful transformation of the E. coli competent cells, for the determination of the success of our fragments’ amplification and purity, after PCR etc.
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Mini-PROTEAN TGX Precast Protein Gels is used for separation of polypeptides ~2–400 kD. It is noted that long-life TGX (Tris-Glycine eXtended) Gels have a novel formulation and can be used for both standard denaturing protein separations as well as native electrophoresis.
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This procedure is based on (Candiano et al Electrophoresis 2004) and has a duration of 5 – 13 hrs. It's used so that the bands that occured for the SDS-PAGE procedure can be stained and observed. This protocol concerns a modified Neuhoff's colloidal Coomassie Blue G-250 stain with considerably higher sensitivity, approaching the one of conventional silver staining.
