General Protocols

• Preparation of general necessities - Various stocks of anticiotics, growth media, stock solutions and other basic necessities have to be available when working in the lab. This protocol describes the way we prepared them.
• PCR Amplification - Amplification of DNA can be done using PCR in a very easy way. With the use of a thermal cycler, template DNA and designed primers, the template can be amplified in only a couple of hours. This saves a lot of time and the product DNA can be used for transformations right away.
• Small Culturing - Cultures of competent cells need to be prepared before protein expression is possible.
• Colony picking - To evaluate if the inserted fragments are of the correct length, colony picking and colony PCR has to be done.
• Colony PCR - To evaluate if the inserted fragments are of the correct length, colony picking and colony PCR has to be done.
• Miniprepping - Miniprepping of the bacteria is needed to obtain the plasmid DNA.
• NanoDrop - To determine the concentration of DNA samples, nanodropping is done.
• Agarose Gel Electrophoresis - To be able to evaluate the length of DNA samples, an agarose gel is prepared.
• Preparing Glycerol Stocks - In order to save the competent cells with the right vectors for later usage, they can be stored in glycerol stocks. These stocks are easy to use when cells are needed at a later moment in your project.
• Plating - To amplify bacteria, plating is required.
• Sequencing - When DNA is amplified and tranformed into competent cells, you want to know wheter the DNA is correct. Using template DNA and specific designed primers, the sequence of vectors can be analyzed.
• PCR Purification - PCR purifaction is for the purification of the product obtained from a PCR reaction. With the QIAquick PCR Purification Kit, products up to 10 ng can be purified. This means that oligos varying from 100 bp to 10 kb can be purified with the simple bind-wash-elute procedure.
• PCR Gel Purification - PCR gel purifaction is for the purification of the product obtained from a PCR reaction. With the QIAquick PCR Gel Purification Kit, oligos varying from 70 bp to 10 kb can be purified with the simply running a gel and a bind-wash-elute procedure.
• Streaking Glycerol Stock - After storing competent cells in glycerol stocks, they can be prepared for usage by means of streaking them on agar plates.

Traditional Cloning & BioBricking

Traditional cloning remains the workhorse of DNA recombinant technology as it is cheap and effective. It has been characterized by the use of restriction enzymes which yield sticky ends. These sticky ends can be ligated to each other by a ligase. The ligated plasmid can subsequently be transformed. We used traditional cloning on and off as well as as a back-up plan if our Gibson Assemblies failed. Figure 1: Overview of the workflow of Traditional Cloning. Traditional Cloning uses restriction enzymes (the scissors) to cut DNA at specific places. Cutting the DNA yields distinct sticky ends which can be ligated together. By cutting both the to be inserted fragment as well as the vector, one can insert the fragment into a vector. As a result, one can obtain a new plasmid.

• Digestion
• Ligation

Transformations

Transformation refers to the act of introducing new plasmid DNA into bacteria. The plasmids can be introduced in bacteria in numerous ways. Perhaps the most well-known methods are electroporation and heat shocking of competent cells. In our project, we used competent cells exclusively. These cells have been chemically modified to transform efficiently. Competent cells can be directly ordered from a wide range of life sciences companies. We used BL21(DE3), NovaBlue, NEB 5-alpha and XL10-Gold (ultra)competent cells exclusively. Figure 2: Transformation is a term used for the introduction of new plasmids into bacteria. Transformation can have place in numerous ways. Often, the term is used intechangeably with transfection. This latter term is, however, reserved for the introduction of plasmid DNA into eukaryotic cells.

• Transformation into NovaBlue - Nova Blue cells can be used for plasmid amplification.
• Transformation into BL21DE3 - BL21DE3 cells are used exclusively for protein expression, as the strain had been optimized (it is a T7 strain, which is the inducable promotor our pETDuet-1 vector has).
• Double transformation - A double transformation can be used to introduce multiple plasmids into competent cells within a single heatshock. In comparison to a singular transformation, the amount of DNA used in a double transformation is far greater. This protocol has been adapted from iGEM TU Eindhoven 2014. To obtain more colonies, the amount of DNA used in a double transformation was increased.

Gibson Assembly

Gibson Assembly is a one-pot assembly method. It requires a linearized vector as well as dsDNA fragments. Linearization of the vector can be realized by the use of restriction enzymes or through PCR. The dsDNA fragments can be obtained through PCR or they can be ordered directly from a manufacturer. In our project, we linearized the vector through PCR and ordered dsDNA fragments directly from IDT. Figure 3: General workflow of Gibson Assembly. The first step consists of linearizing the vector using either PCR or digestion by restriction enzymes. Next, the linearized vector and dsDNA fragments are introduced in a tube with the Gibson Assembly Master Mix, and incubated at 50°. The resulting mixture is transformed into competent cells and analyzed using colony PCR to select the correctly assembled vectors.

• Vector Linearization - A linear vector is a prerequisite for Gibson Assembly. Linearization can be realized through restriction or through PCR. In our protocol, we use PCR as this yields scarless constructs. This protocol consists of a PCR step, an optional DpnI digestion step, an optional PCR purification step, a NanoDrop step and an optional gel electrophoresis step.
• NEBuilder HiFi Assembly - During our iGEM summer, we used the NEBuilder HiFi Assembly Kits. These kits contain a high-fidelity polymerase rather than a normal polymerase, limiting the occurence of errors during the Gibson Assembly. This protocol contains the one-pot assembly method as well as transformation of the product into NEB 5-alpha cells.

Non-natural protein expression & FACS

The Fluorescence-Assisted Cell Sorter will be used to determine whether the click reaction occurs. To enable the click reaction, proteins have to be expressed with the unnatural amino acid. The proteins expressing this unnatural amino acid can subsequently be incubated with DBCO-functionalized TAMRA dye to enable quantitative measurement of the click reaction. Figure 4: To verify whether the click reaction has occured, we incubate the cells with DBCO-functionalized TAMRA. If the outer membrane protein is functionalized with the non-natural amino acid, this TAMRA dye binds to the membrane proteins covalently. In that case, the cells will remain fluorescent after a few washing steps.

• Preparation of FACS samples
• Protein expression with the non-natural amino acid

Alginate Beads

• Making alginate beads - Bacteria can be placed in an controlled environment using alginate beads

Testing the COMBs

• SDS-Page Analysis - A SDS-page gel is a useful to check if your proteins are expressed.
• Complementary DNA assay - Bringing them together with complementary DNA.
• Bioluminescence & Fluorescence assay - One can measure bioluminiscence of the luciferases (NanoLuc & NanoBit) for verifying its functionality. One can measure the fluorescence of the fluorophore (mNeongreen). When both NanoLuc & mNeongreen are present in the cell BRET can occur & be measured as well.