Experiment Overview

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To make the Rh(D) antigen ineffective, we use Bioinformatics Engineering to redesign the Anti-D antibody into smaller, more efficient forms such as a Single-chain variable fragment (scFv) and Single-domain antibody (sdAb). Moreover, we design a conversion kit to make the process of blocking Rh(D) antigen more convenient.

Cloning (Subcloning)

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Vector:

We will be using pET 28a as the vector. The pET-28a vector is a widely used plasmid in molecular biology, particularly for the expression of recombinant proteins in Escherichia coli (E. coli). It is part of the pET (plasmid for expression by T7 RNA polymerase) system, which is known for its high levels of protein expression.

Restriction Digest of Plasmid DNA:

In our project, the DNA sequences we originally ordered were cloned into the pUCIDT (Amp) vector from IDT, which contains an ampicillin resistance selection marker. To extract our desired sequences, we used the restriction enzymes XhoI and NdeI. These enzymes specifically recognize their respective sites within the multiple cloning region of the vector. We also cut the pET-28a vector, which we are using for cloning, with the same restriction enzymes. By performing a double digestion with XhoI and NdeI on both the pUCIDT vector and the pET-28a vector, we can cut out the target DNA sequences for further cloning, enabling downstream protein expression.

Restriction Enzymes: Nde1(CATATG)/Xho1(CTCGAG):

NdeI is a restriction enzyme frequently used in molecular biology, particularly in DNA cloning. It recognizes and cuts the palindromic DNA sequence 5’- CATATG - 3’. This cutting occurs between the C and A nucleotides, producing sticky ends with a 5' overhang, which facilitates the ligation of DNA fragments with compatible ends. NdeI is especially useful for opening cloning vectors like pET-28a, where an NdeI site is present upstream of the target gene insertion point within the multiple cloning site. This makes it ideal for inserting genes that require compatible ends for expression. The enzyme functions optimally at 37°C in the appropriate buffer provided by the manufacturer.

XhoI is a widely used restriction enzyme in molecular biology, especially for cloning and genetic manipulation. It recognizes the palindromic DNA sequence 5’- CTCGAG - 3’ and cleaves between the C and T nucleotides, producing sticky ends with 5' overhangs. These overhangs facilitate the ligation of DNA fragments with complementary ends, making XhoI useful in recombinant DNA technology. It is often employed in conjunction with other restriction enzymes to cut and insert genes into plasmid vectors. The enzyme operates optimally at 37°C in its appropriate reaction buffer, and its sticky ends allow efficient recombination with compatible sequences. It is commonly used alongside vectors like pET-28a, where an XhoI site is present in the multiple cloning sites, enabling the insertion of target genes for protein expression or other purposes.

Experimental Steps for Digestion:

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1. Prepare the digestion reaction mix:

We start with the DNA plasmid (pUCIDT with the desired insert), using about 1-2 µg.

We add 1 µL of XhoI enzyme (typically 10 units/µL).

Next, we include 1 µL of NdeI enzyme (also typically 10 units/µL).

Then, we add 5 µL of 10X restriction enzyme buffer, using the buffer compatible with both enzymes, usually NEBuffer 4.

Finally, we add nuclease-free water to bring the total volume to 50 µL.

2. Cut the pET-28a vector:

We prepare a similar reaction mix for the pET-28a vector, using the same amounts of enzymes and buffer.

3. Incubation:

We mix the reaction components gently and incubate both reactions at 37°C for 1-2 hours to allow the enzymes to cut the DNA.

4. Heat inactivation:

After digestion, we heat both reactions at 65°C for 20 minutes to inactivate the enzymes, following the manufacturer’s instructions.

Ligation

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After successfully digesting both the pUCIDT vector with our desired insert and the pET-28a vector, we proceed with the ligation process. In this step, we mix the cut pET-28a vector with the purified insert from the pUCIDT vector in the presence of DNA ligase, which catalyzes the formation of phosphodiester bonds between the compatible sticky ends of the DNA fragments. We carefully optimize the molar ratio of vector to insert, typically aiming for a ratio of 1:3, to enhance the likelihood of successful ligation. The reaction is incubated at room temperature or at 16°C overnight, allowing sufficient time for the ligase to join the fragments together. Once ligation is complete, we can transform the ligated product into competent E. coli cells for propagation and expression of our target protein.

Transforming

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After completing the ligation of our desired insert into the pET-28a vector, we are ready to transform the plasmid into competent E. coli B cells. Transformation is a critical step in molecular cloning, allowing us to introduce the recombinant plasmid into this specific bacterial host for propagation and expression. E. coli B is known for its ability to efficiently replicate plasmids, making it an ideal choice for our experiments. This step will enable us to select for bacteria that have successfully taken up the plasmid, facilitating further analysis and protein expression in subsequent experiments. Through this process, we aim to generate a culture of transformed E. coli B that carries our recombinant plasmid, allowing us to produce our target protein effectively.

Escherichia coli B

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E. coli B is a strain of the bacterium Escherichia coli that is widely used in molecular biology and biotechnology. Known for its rapid growth and ability to replicate plasmids efficiently, E. coli B serves as an essential host for the expression of recombinant proteins and the production of DNA. This strain is particularly valued for its genetic stability and ease of manipulation, making it suitable for various applications, including cloning, gene expression, and genetic engineering. Its ability to take up foreign DNA through transformation allows researchers to explore genetic functions, produce proteins, and study biological processes in a controlled laboratory setting. The robustness and reliability of E. coli B make it a foundational tool in molecular biology research. To transform our plasmid into competent E. coli B cells, we begin by thawing the competent cells on ice, handling them gently to maintain their viability. Once thawed, we add the ligated plasmid DNA to the cells, typically using a small volume, such as 1-5 µL. We gently mix the solution by flicking the tube to avoid disrupting the cells. Next, we incubate the mixture on ice for about 30 minutes to allow the plasmid DNA to adsorb to the cell surface, which is crucial for maximizing the efficiency of transformation. After this ice incubation, we subject the cells to a brief heat shock by placing them in a water bath at 42°C for approximately 45-60 seconds. This heat shock creates a thermal imbalance that helps facilitate the uptake of DNA into the cells. Immediately following the heat shock, we return the cells to ice for another 2 minutes to stabilize them. We then add recovery medium, such as SOC or LB broth, to the transformed cells and incubate them overnight at 37°C with shaking. This extended recovery period allows the cells to express the antibiotic resistance gene carried by the plasmid effectively.

Plating

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After the overnight incubation, we plate the transformed cells onto LB agar plates containing the appropriate antibiotic, such as ampicillin, to select for those that have successfully taken up the plasmid. We then incubate the plates at 37°C for about 16-24 hours. The following day, we checked for the presence of colonies, ⅝ successfully grew colonies. The colonies that grow on the selective medium are those E. coli B cells that have integrated the plasmid, allowing us to move forward with further analysis and experimentation on the recombinant DNA.

IPTG Induction

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IPTG induction is a common technique used in molecular biology to activate the expression of genes cloned into plasmids that contain the lac operon system. IPTG (isopropyl β-D-1-thiogalactopyranoside) is a non-hydrolyzable analog of lactose that can bind to the lac repressor protein. When IPTG is added to a culture of E. coli containing a plasmid with a lac promoter, it binds to the repressor and causes a conformational change, effectively releasing the repression of the promoter. This allows RNA polymerase to bind and initiate transcription of the downstream gene, leading to the production of the target protein. IPTG induction is particularly useful because it allows for tight control over gene expression. By adding IPTG at specific concentrations and times, researchers can optimize the expression levels of their target proteins, minimizing potential toxicity or metabolic burden on the bacterial cells. This method is widely used in protein production, especially for recombinant proteins. In our experiment, we divide the transformed E. coli B cells into two groups to compare protein expression levels: one group undergoes IPTG induction while the other group remains uninduced. For the induced group, we add IPTG to the culture at a specific concentration to activate the expression of the target protein encoded by our plasmid. This allows us to assess how effectively the induction enhances protein production. In contrast, the uninduced group serves as a control, enabling us to determine the baseline expression levels without IPTG. By analyzing the protein yields from both groups, we can evaluate the impact of IPTG induction on the expression of our recombinant protein, providing insights into the efficiency of the lac operon system in our experimental setup.

Protein Purification

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To isolate the recombinant Anti-D antibody from the E. coli lysate, inclusion body purification method using Ni-NTA His•Bind resin was performed. This resin specifically binds to the His-tag fused to the antibody, allowing for its selective capture. The purified protein was then dialyzed to remove impurities and concentrate the sample. Finally, the concentrated protein was stored at -20°C for long-term preservation.
1. Process the induced culture according to steps 1–4 above for the soluble protein fraction.
2. Resuspend the pellet from step 4 above in the same volume of BugBuster reagent that was used to suspend the cell pellet. Pipet up and down and vortex to obtain an even suspension (see note i below).
3. Add lysozyme to a final concentration of 200 µg/ml (use 1/50 volume of a freshly prepared 10 mg/ml stock in water). Vortex to mix and incubate at room temperature for 5 min.
4. Add 6 volumes of 1:10 diluted BugBuster reagent (in deionized water) to the suspension and vortex for 1 min.
5. Centrifuge the suspension at 16,000 × g for 15 min at 4°C to collect the inclusion bodies. Remove the supernatant with a pipet.
6. Resuspend the inclusion bodies in ½ the original culture volume of 1:10 diluted BugBuster, mix by vortexing, and centrifuge as in step 5. Repeat this wash step 2 more times.
7. Resuspend the final pellet of purified inclusion bodies in your buffer of choice. Inclusion bodies prepared in this manner are compatible with resuspension in 1X IB Solubilization Buffer provided in Novagen’s Protein Refolding Kit.