Theoretical Task:

Design a gene. Describe a detailed workflow for constructing and expressing it. Identify how the parts of your genetic construct relate to DNA replication and the Central Dogma of Molecular Biology

 

 

Firstly, we must have clear the goal of The central dogma of biology. This explains the flow of genetic information, from DNA to RNA, to make a functional product, a protein.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                             This figure aim to present the key feature of average gen.

 

  I inevitably miss many interesting features such as overlapping genes, alternative splicing,trans-splicing, or the subcellular locations of events.

 

   Despite these concessions, the diagrams provide an accurate overview of the important common aspects of gene structure necessary for understanding genetics.

 

   Although DNA is a double stranded molecule, typically only one of the strands encodes information that the RNA polymerase reads to produce protein-coding mRNA or non-coding RNA.

 

   This 'sense' or 'coding' strand, runs in the 5 'to 3' direction, the open reading frame (ORF) of a gene is therefore usually represented as an arrow indicating the direction in which the sense strand is read.

 

 

   The promoter is located at the 5 'end of the gene and is composed of the core promoter sequence and the proximal promoter sequence. The core promoter marks the start site for transcription by binding RNA polymerase and other proteins necessary for copying DNA to RNA. The proximal promoter region binds transcription factors to modify the affinity of the promoter for RNA polymerase.

 

   An additional layer of regulation occurs for protein coding genes after the mRNA has been processed to prepare for protein translation. Only the region between the start and stop codons encodes the final protein product. The flanking untranslated regions (UTRs) contain further regulatory sequences. The 3 'UTR contains the terminator sequence, which marks the endpoint for transcription and releases the RNA polymerase. The 5 'UTR binds the ribosome, which translates the protein-coding region into a string of amino acids that fold to the final protein product. In the case of genes for non-coding RNAs the RNA is not translated but instead folds to be directly functional Eukaryotic genes typically have more regulatory elements to control gene expression compared to prokaryotes.

 

    The key feature of the structure of eukaryotic genes is that their transcripts are typically subdivided into exon and intron regions. Exon regions are retained in the final mature mRNA molecule, while intron regions are spliced out (excised) during post-transcriptional processing.Indeed, the intron regions of a gene may be considerably longer than the exon regions. Once spliced together, the exons form a single continuous protein-coding regions, and the splice boundaries are not detectable. Eukaryotic post-transcriptional processing also adds a 5'-cap to the start of the mRNA and a poly-adenosine tail to the end of the mRNA. These additions stabilize the mRNA and direct its transport from the nucleus to the cytoplasm.

 

 

Lab Task:

 

In this assignment, we had the experience building genes and expressing them in bacteria.

 

To do this, was important understand about the amplifying DNA, Golden Gate Assembly, how setting electrophoresis gel and agar plates.

 

To start, it is necessary to take into account the materials and reagents needed and understand how they work so that the test is a success and not just follow the instructions.

 

In the lab task to construct a blue fluorescent protein from a Green fluorescent protein We begin by dividing the practice into three phases:

Phase 1: we prepare the Golden Gate fragments from complementarity oligos pairs, for to do this we first prepared 3 tubes that contained the oligos pairs (T500; OR2OR1Pr and 5UTR) this we mixed with T7 DNA ligase buffer, T4 PNK and water.

The DNA ligase buffer catalyze the formation of a phosphodiester bond between adjacent 5´ phosphate and 3´ hydroxyl groups of duplex DNA.

The T4 PNK Catalyzes the transfer and exchange of Pi from the γ position of ATP to the 5´ -hydroxyl terminus of polynucleotides (double-and single-stranded DNA and RNA) and nucleoside 3´-monophosphates to allow subsequent ligation. Polynucleotide Kinase also catalyzes the removal of 3´-phosphoryl groups.

We incubated the samples at 37°C for 1 hr, then heat mixtures at 95°C for 5 min and gradually cool to RT for 1hr.

Phase 2: we prepare the Golden Gate fragments by PCR, for that we prepared 5 tubes for PCR and first we mixed the primers with the complementary primers, each tubes contained Template Y, dNTPS, Phusion polymerase buffer, Phusion DNA polymerase and water.

Template Y, is a nucleotide sequence that directs the synthesis of a sequence complementary to it by the rules of Watson crick base pairing. That is to say a molecule that provides the structural mold to create similar molecules.

dNTPs (deoxynucleotide triphosphates) are the monomeric substrates for the polymerization reaction. It is a mixture of the four monomeric units, dATP, dTTP, dCTP and dGTP that will ultimately make up the DNA that is polymerized during PCR. Each of the dNTP molecules contains the the DNA base in a highly energized triphosphate form. Each base is added to the DNA strand thru a phosphodiester bond (a phosphate group between each base) and a molecule of pyrophosphate (two phosphates bound to each other) is released, providing the energy for the polymerization reaction.

Phusion polymerase buffer providing a suitable chemical environment for optimum activity and stability of the DNA polymerase

Phusion DNA Polymerase possesses 5´→ 3´ polymerase activity, 3´→ 5´ exonuclease activity and will generate blunt-ended products. High-Fidelity DNA Polymerases are important for applications in which the DNA sequence needs to be correct after amplification.

We ran the following program PCR in the thermocycler, this consisted of a series of 30 repeated cycles, with each cycle consisting of three discrete temperature steps. The individual steps common to most PCR methods were as follows:

Stage 1:

 Initial: This step is only required for DNA polymerases that require heat activation by hot-start PCR. It consisted of heating the reaction chamber to a temperature of 98°C for 30 sec.

Stage 2:

• Denaturation: This step is the first regular cycling event and consisted of heating the reaction chamber to 98 °C for 10 sec. This causes DNA melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules

• Annealing: In the next step, the reaction temperature was lowered to 60 °C for 30 seconds, allowing annealing of the primers to each of the single-stranded DNA templates. Two different primers are typically included in the reaction mixture: one for each of the two single-stranded complements containing the target region. During this step, the polymerase binds to the primer-template hybrid and begins DNA formation.

• Extension/elongation: The temperature at this step depends on the DNA polymerase used; in this case we used a temperature of 72 °C  with this enzyme for 30 sec. In this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture that are complementary to the template in the 5'-to-3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxy group at the end of the nascent (elongating) DNA strand.

Stage 3:

• Final elongation: This single step was performed at a temperature of 72 °C  for 5 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA was fully elongated.

We ran the finished PCR reaction on 1% agarose TAE gel, and and purified the correct size band with a Qiagen gel extraction kit for later use in phase 3.

Phase 3:  Combine and assemble the Golden Gate fragments and heat shock Golden Gate assembly into chemically competent E.coli cells.

For each fragment from phase 2 and 3 were mixed with the following in a thermocycler tube with cutsmart buffer, ATP, T4 DNA ligase, Bbsl-HF and Water.

The cutsmart buffer improve the performance for optimum activity of BbsI-HF that recognizes a specific, short nucleotide sequence and cuts the DNA only at that specific site.

The ATP provides energy in the reactions and the T4 DNA ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA and some DNA/RNA hybrids. T4 DNA ligase will seal nicks for these DNA substrates.

We ran again the following assembly program in the thermocycler, the stage 1 for 30 cycles, the digest at 37°C for 5 min and the Ligate at 16°C for 5 min and the Stage 2 at 55°C for 10min.

FINALLY, we did the heat shock Golden Assembly into chemically competente E.coli cells.

For that, Thawed pre-aliquoted 25 ul E coli cells on ice for 30 min, then added 2 ul of assembly to cells and keeped on ice for 15 min, we heated shock cells at 42C for 45 sec and immediately placed on ice and mixed 500 ul outgrowth media with cells and plated 100 ul.