Part:BBa_K5490017:Design

Structural Design and Experiments

Introduction to the Project

When performing immunocytochemistry on the original plasmid pCMV-MycNFAT provided by Professor Meško and her team, significant background noise was observed. In contrast, the production of NFAT was successfully detected via Western blot analysis. To address the background issue, we decided to add a FLAG tag to the N-terminus of the Myc tag through subcloning. For this purpose, we utilized the pCMV-FLAG-TRIM32 construct (BBa_K5490034), which was readily available in the lab. To facilitate the subcloning process, it was necessary to remove the TRIM32 sequence downstream of the FLAG tag while extracting the NFAT insert from the original construct. We needed to identify suitable enzymes to maintain directionality and preserve the open reading frame (ORF). We developed two different strategies to achieve this outcome.

Insert Preparation:

We identified a HindIII site upstream of the NFAT insert, which will be used for blunt-end cloning, and an XbaI site downstream to ensure directionality.

Vector Preparation:

For the vector, we located an XhoI site upstream of the TRIM32 gene for blunt-end cloning and an XbaI site downstream to maintain directionality.

Insert Preparation:

We first identified a BglII site at the borders of the NFAT insert; however, an additional BglII site was present within the NFAT sequence, necessitating a partial digestion.

Vector Preparation:

For the vector, we identified a BamHI site at the borders of the TRIM32 gene. BamHI is isoschizomeric to BglII, but it cuts within the TRIM32 gene, which is acceptable as we only require the backbone from this construct.

Building a new composite part

Preparation of the Insert:

First, we linearized the construct using HindIII, followed by treatment with the Klenow fragment to convert the sticky ends into blunt ends. We then digested the linearized product with XbaI and isolated the insert through gel extraction.

Preparation of the Vector:

We linearized the vector with XhoI, converted the sticky ends to blunt ends using the Klenow fragment, performed another digestion with XbaI, and isolated the backbone via gel extraction.

Preparation of the Insert:

Initially, we linearized the pCMV-MycNFAT construct with XbaI, then performed partial digestion with BglII at four different time points: 10, 20, 30, and 45 minutes. We identified the correct band through gel electrophoresis and isolated it via gel extraction.

Preparation of the Vector:

Complete digestion of the vector was carried out using BamHI, followed by isolation of the vector and subsequent digestion with XbaI. The backbone fragment was then removed through gel extraction.

Ligation Step

For the ligation we mixed the insert and vector in a 3:1 molar ratio and used T4 ligase. Notably, for the partial digestion strategy, we employed a buffer specialized for blunt-end ligation. The constructs were then amplified in DH5α cell lines.

Testing of another tagged version of the synthetic NFAT

Unfortunately, in the first scenario using the Klenow fragment, only a single colony was produced. Upon screening via restriction digest analysis, this colony did not yield the desired cutting pattern when viewed on a gel. In contrast, in the second scenario, multiple colonies were obtained, and suitable colonies were identified through restriction digest analysis. A midi prep was subsequently conducted to extract sufficient plasmid quantities, with measurements performed using software that analyzed light intensity from the gel, alongside NanoDrop readings.

Learning from a successful cloning

One important lesson learned during this project is that not all antibody tags are suitable for performing immunocytochemistry, as their effectiveness can vary depending on the specific protein being studied. Additionally, we gained valuable experience in subcloning techniques and realized that multiple strategies can achieve the same goal, which is beneficial since one method may not always be successful. This adaptability is crucial in molecular cloning, as it increases the likelihood of obtaining the desired constructs.

• 1.pCMV-FlagNFAT(BglII/BamHIxXbaI)-22-XhoI
• 2.blank well
• 3.pCMV-FlagNFAT(BglII/BamHIxXbaI)-22-PciI
• 4.pCMV-FlagNFAT(BglII/BamHIxXbaI)-22-HindIII
• 5.pCMV-FlagNFAT(BglII/BamHIxXbaI)-22-supercoiled
• 6.1kb ladder plus by NEB

The NFAT construct was kindly provided by Benčina M, with the goal of studying its translocation to the nucleus after ionophore stimulation. For detection, we used an immunohistochemical approach targeting the Myc-tag epitope, which was fused to the N-terminus of the NFAT construct. However, the antibody against the Myc-tag epitope generated significant background noise. While it had some affinity for its target, it also bound nonspecifically to other molecules, leading to an inconclusive image under the microscope. Despite this issue, we confirmed the expression of the synthetic protein via Western blot, which motivated us to explore alternatives. Specifically, we decided to add a Flag tag in front of the Myc-tag epitope, leading us to try two different cloning strategies to achieve this.

First, we attempted to fuse the Flag tag using subcloning. In this approach, we extracted the insert and blunt-ended one side of both the vector and the insert while keeping the other side sticky to maintain directionality during the ligation step. Unfortunately, this strategy was unsuccessful. The second strategy, which worked, involved performing a partial digestion, as one of the restriction enzyme sites was located within the NFAT insert itself. We then performed directional cloning into a new vector containing the Flag tag .