Synthetic Biology and Biological Circuits Assignment | Essay help Services

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Final lab: Synthetic Biology and Biological Circuit labs Your final paper should be about 8-10 pages long, double-spaced, font size 12. In your final paper you must answer all of the following questions (in this exact order). Clearly mark each section (Introduction, Materials and Methods, Results/Discussion). 2 Sources https://blog.addgene.org/plasmids-101-gateway-cloning https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/gibson-assembly

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  • Welcome to the Biological Circuit Lab
  • In this lab you will design a biological circuit to sense and destroy cancer cells. This lab build upon the theory learned in the Synthetic Biology labs and focuses on the Gibson assembly technique that can be used to construct complex biological circuits.
  • Following is a list of the most important theory pages:

·         Biological circuits

  • Biological circuits consist ofbiological parts that perform logical functions, analogous to an electronic circuit. Biological circuit engineering builds upon the basic mechanics of gene expression and regulation. The enormous complexity of biological systems remains a huge challenge that often leads to unpredictable outcomes.
  • Biological circuit partsare recombinant DNA sequences, which by themselves are simply a piece of code without a function. If circuit parts are transformed into a host organism, this code will be translated into a specific protein with a specific function. Biological circuits are assembled using molecular cloning  Recent research has developed standardized techniques to build circuit libraries that can be efficiently recombined to create novel circuits.
  • Molecular cloning

Molecular cloning refers to the assembly of DNA molecules into a vector and the subsequent transformation of an organism (often bacteria or yeast). Molecular cloning methods are central to biology and medicine. The term molecular cloning comprises many different techinques. A typical molecular cloning flow looks as follows:

  1. Isolation of the gene of interest
  2. Cloning the gene into a vector
  3. Transforming the host organism with the vector construct
  4. Antibiotic selection of transformed cells
  5. Isolation of clones with the same genetic background

Confirmation of plasmid assembly can be determined by performing DNA sequencing. It is important to confirm that the inserts have been successfully ligated into a plasmid vector with the correct conformation and reading frame. Frame shift can cause nonsense or misense mutations that lead to the expression of nonfunctional proteins.

·         Cloning techniques

Many molecular cloning methods have been established to produce recombinant DNA. Oftentimes several different approaches will work for any clonig project. Following are some well established methods:

 

 

·         Restriction enzymes

Restriction enzymes cleave the sugar-phosphate backbone of double-stranded DNA. They recognize a specific site of double-stranded DNA and cleave it within, or adjacent to, their recognition site. Restriction enzymes are a very important tool in molecular biology. They allow us to cut DNA strands in a highly predictable manner.

The resulting ends are divided into:

  • Sticky ends: One strand is longer than the other, resulting in either a 3′ or 5′ overhang.
  • Blunt ends: Both strands are cut at the same base pair, resulting in an end without an overhang.

Following are two examples of restriction enzymes

XbaI (pronounced Xba-one): produces sticky ends. It recognizes the following restriction site:

5′—T CTAGA—3′

3′—AGATC T—5′

I-SceI (pronounced Sce-one): has a very unique restriction site that does not naturally occur in mice or human genomes. Hence, it is very useful for highly specific restrictions. SceI recognizes the following restriction site:

5’…TAGGGATAA CAGGGTAAT…3′

3’…ATCCC TATTGTCCCATTA…5′

 

·         Types of restriction enzymes

Restriction enzymes can be divided into 4 groups based on their recognition sequence, subunit composition, cleavage position, and cofactor requirements.

Following is a list of the four types of restriction enzymes:

Type I restriction enzymes are compromised of one enzyme with different subunits for recognition, cleavage, and methylation. This enzyme recognizes and methylates at the same sequence but cleaves DNA up to 1000 base pairs away from the initial recognition site.

Type II restriction enzymes are compromised of two different enzymes, which cleave or modify the recognition sequence.

Type III restriction enzymes consist of one enzyme with two different subunits, each for recognition and modification or cleavage. This enzyme recognizes and methylates at the same sequence but cleaves 24-26 base pairs away from the initial recognition site.

Type IIs restriction enzymes are compromised of two different enzymes with asymmetrical recognition sequence. Cleavage occurs on one side of recognition site up to 20 base pairs away.

·         Restriction condition

Most of the restriction enzymes have an optimum temperature of 37°C and an optimum pH of pH 7.2 or pH 8.5. These physical environment requirements must be met to ensure that the enzyme reaches its maximum activity. Incubation temperatures affect the activity of restriction enzymes greatly. The enzyme can denature outside the desirable physical condition range. Enzymes work very specifically; therefore, they will not work properly if their structure deteriorates. As each restriction enzyme is isolated from different bacteria and have different optimum reaction conditions, they require specific buffers for each restriction reaction.

Table 1: Enzyme activities. The number for each buffer sigifies the activity in the respective enzyme. If there is a * the enzymes may exhibit star activity in this buffer.

Enzyme Sequence Buffer1 Buffer2 Buffer3 Incu. Temp.
EcoRI G’AATTC 25 100* 50 37°C
KpnI GGTAC’C 100 75 10 37°C
PstI CTGCA’G 75 75 100 37°C
SpeI A’CTAGT 75 100 25 37°C
XbaI T’CTAGA 10 100 75 37°C
XhoI C’TCGAG 75 100 100 37°C

 

·         Gibson assembly

Gibson assembly is a cloning technique that does not rely on restriction sites like traditional cloning techniques. The Gibson assembly relies on the presence of homologous regions in the ends of the DNA pieces.

The three enzymes T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase all process different parts of the DNA ends. First, the T5 exonuclease, chews back on the 5’ ends, creating single-stranded complementary overhangs between the two inserts. These overhangs anneal and the Phusion polymerase inserts the missing nucleotides between the sequence of double stranded DNA, using the homologous overhang as priming sequence. Once started, the Phusion reaction is much faster than the slow nuclease and the gaps are quickly filled. As the last step the ligase closes the strand break and fuses the two DNA fragments together. All these enzymes are active at 50 ºC. As a result, multiple pieces of DNA can be assembled in a single tube.

The Gibson assembly is used to construct large circuits from position vectors.

Read more about how to set up a Gibson reaction.

·         Position vector

Position vectors are specialized destination vectors that can be efficiently used to Gibson assemble large biological circuits. Each position vector contains a chromatin insulator sequence, a Gateway recombination cassette, and a polyadenylation sequence.

These DNA motifs are flanked by two different, 40 base pair long, unique nucleotide sequences (UNS). The UNSs are flanked in turn by two I-SceI restriction sites. This arrangement of DNA motifs allows for fast and reproducible assembly of large biological circuits.

Entry vectors with circuit parts can be assembled into the position vector using the gateway cloning technique. SceI digestion of the resulting plasmid will yield a fragment with the circuit parts and the afore mentioned DNA motifs, flanked by the two UNSs. These fragments can be used for Gibson assembly right away. The uniqueness of the UNSs ensure specific assembly of a large number of different fragments.

The following steps are necessary to assemble a large circuit from position vectors:

 

 

·         Welcome to the Synthetic Biology Lab

  • In this lab you will design a biological circuit to sense and destroy cancer cells. You will use the Gateway cloning technique to assemble different circuit parts and transform bacteria cells with the vector construct.
  • Following is a list of relevant theory pages:

·         Biological circuits

Biological circuits consist of biological parts that perform logical functions, analogous to an electronic circuit. Biological circuit engineering builds upon the basic mechanics of gene expression and regulation. The enormous complexity of biological systems remains a huge challenge that often leads to unpredictable outcomes.

Biological circuit parts are recombinant DNA sequences, which by themselves are simply a piece of code without a function. If circuit parts are transformed into a host organism, this code will be translated into a specific protein with a specific function. Biological circuits are assembled using molecular cloning techniques. Recent research has developed standardized techniques to build circuit libraries that can be efficiently recombined to create novel circuits.

·         Gateway cloning

The Gateway technology provides a fast and efficient route for cloning. This technology relies on the use of modified versions of the recombinases from the bacteriophage lambda. This virus inserts its genome into the host DNA using these enzymes. The Gateway cloning system uses them to achieve high efficiency, site specific recombination. The second useful characteristic of this system is its recognition sites for the clonase enzymes called att sites, and the use of different types of vectors.

Watch the animation below to get an overview of the Gateway reaction.

BP and LR reactions

BP and LR reactions are the way different DNA segments are moved from one place to another within the constructs. During the BP reaction, attB and attP sites are recombined. This reaction swaps the DNA between strands, creating an attL and an attR site. During the LR reaction, attL and attR sites are similarly recombined, yielding attB and attP sites. There is a limited number of att sites, annotated with numbers. This means that there is a limited number of combinations. Each site only recombines within the group: for example, attL1 sites will only recombine with attR1 sites, yielding attB1 and attP1 sites. The orientation of these sites is also specific, and the gene construct highly predictable. The desired construct can be built by selecting for the proper att ends, the reactions, and the vectors.

Vector types

The recyclable att sites are ideally suited for creating vector libraries that can be used to efficiently combine different circuit parts.

  • Expression vector(AmpR): The expression clone is the final product of a gateway reaction. It is the complete plasmid, ready for transformation.
  • Donor vector(ccdB, KanR): The donor vector provides the backbone for the creation of entry clones. Donor vectors are typically denoted with names starting with pDONR. These plasmids carry attB or attP sites flanking the ccdB gene, as well as the kanamycin resistance gene.
  • Entry vector(KanR): The entry vectors are created from the donor vector and a DNA sequence flanked with the matching att sites. These sequences of interest are usually produced by PCR, with primers that contain the att sites. Entry vectors contain attL or attR sites flanking the sequence of interest, as well as the kanamycin resistance gene. The entry vectors are ideally suited for a library of circuit parts.
  • Destination Vector(ccdB, AmpR): The destination vector provides the backbone for expression clones. Destination vectors are typically denoted with names starting with pDEST. These plasmids contain the ccdB gene flanked by attL or attR sites, as well as an ampicillin resistance gene. Destination vectors also contain the origins of replications for specific hosts and additional DNA motifs.

Selection

The selection of the vectors of interest in the different steps of the Gateway cloning procedure is very important. For selection, the Gateway system relies on two antibiotic resistancesand the ccdB gene for positive selection. The antibiotic resistance enables transformed cells to grow on a medium containing antibiotics that kill untransformed cells.

Click here if you want to learn more about ampicillin resistance..

ccdB encodes a bacteriotoxin that prevents DNA replication. All of the backbones for Gateway clones (pDEST and pDONR) carry this gene, and only bacteria carrying an additional ccdA gene will grow when transformed with these plasmids.

 

·         Antibiotic selection

The number of transformed cells is usually very low compared to the cells that did not take up the vector. Hence, the transformed cells have to be selected somehow. The most common method is based on antibiotic resistance genes that are part of the vector construct. After transformation, the cells can be grown on a rich medium containing the matching antibiotics. On this selective media, only cells containing the plasmid vector are able to multiply and produce colonies.

Antibiotic selection is an important step of molecular cloning.

·         Plasmid miniprep

There are several different methods to purify plasmid DNA from bacterial cells. Miniprep is a rapid, small-scale isolation method, which relies on alkaline lysis of the cells, followed by silica column purification of the DNA.

The following steps have to be performed to purify plasmids from a bacteria culture:

  1. Pelleting the cells by centrifuging them at 10K rpm for 3 min.
  2. Homogenizing the cells by adding a homogenization buffer and pipetting repeatedly.
  3. Lysing the cells with a lysis buffer (containing a detergent) and by inverting the tube repeatedly.
  4. Lysing the cells for a maximum 5 minutes at room temperature.
  5. Stopping the reaction by adding a neutralization buffer, and again inverting the tube several times. White clumps appear, which are the cells’ debris.
  6. Pelleting the debris by centrifuging for 10 min at 13K rpm. In the end, your plasmid will be in the supernatant (the liquid phase).
  7. Applying the supernatant to the silica column and centrifuging at 13K for 1 min. Your plasmid will bind to the filter at the bottom of the column. Discard the flow-through.
  8. Washing your DNA twice with a wash buffer (add, centrifuge, discard).
  9. Centrifuging 1 minute without adding anything, in order to get rid of any residual buffer.
  10. Transferring your column to a clean 1.5ml tube and add elution buffer (water with 10mM Tris_HCl). Leave it on your bench for couple of minutes before centrifuging again.
  11. Checking DNA concentrations in the nano-drop.

·         Gel electrophoresis

Gel electrophoresis is a method to separate charged macromolecules (DNA, RNA, or proteins) of different sizes and to estimate their length.

Because nucleic acids are negatively charged ions at neutral or basic pH in an aqueous environment, this technique is often used to separate DNA or RNA molecules. This is necessary, for example, in the case of DNA profiling or to study RNA integrity.

Gel electrophoresis is often used to separate PCR amplified DNA fragments. The process is also useful to isolate and extract DNA fragments of a specific size.

In the virtual lab we use the E-gel machine to perform gel electrophoresis (see image below).

 

·         Gel electrophoresis preparation

To perform a gel electrophoresis experiment you need:

Semisolid, porous gel matrix: This is usually an agarose or polyacrylamide gel. In the virtual lab this gel is already prepared inside the E-gel machine.

DNA or RNA sample: Isolated, treated DNA or RNA, of a sufficient quantity to be visible in a gel electrophoresis experiment.

Loading buffer: Loading buffer contains a colour reagent to help visualize how far the DNA or RNA has traveled during gel electrophoresis. Loading buffer also makes the sample heavier, so it will sink to the bottom of the gel.

It typically contains the following chemicals:

  • Coloring reagent, for example, xylene cyanol, cresol red, bromophenol blue, or orange G
  • High viscosity reagent, for example, Ficoll, sucrose, or glycerol to make the sample more viscous and heavier
  • Water to dilute the above mentioned reagents

Dye: Fluorescent or colored dyes are used after the electrohoresis is finished to visualize nucleic acids in the gel matrix.

Molecular weight standard samples: These are run alongside the DNA sample to provide a size reference. They are also known as “ladders”.

 

·         Gel electrophoresis procedure

In gel electrophoresis, a positive electrode is positioned at one end of the gel layer, and a negative anode at the other. The gel layer is formed with small wells in one end, where the DNA fragments and reagent mixture is loaded.

DNA is negatively charged. When a current is passed through the gel, the DNA moves through the pores in the gel towards the positive electrode. Smaller DNA molecules move faster through the gel than larger DNA molecules, leading to size separation. This difference in the rate of migration separates the fragments on the basis of size.

After electrophoresis, DNA is visualized and appears as ‘bands’ of grouped DNA fragments of the same length. Only a large amount of DNA (millions of copies) can be visualized with this method.

The sizes of the DNA samples can be estimated by comparing the distance with the DNA ladder (‘marker DNA’ in Fig.1).

Figure 1. Fragments of different lengths from a PCR reaction are run on a gel. The fragments will move at a speed and a distance relative to their size: smaller DNA molecules will move in the gel faster and further than longer DNA molecules.

·
Gel electrophoresis analysis

After electrophoresis, the different fragments are visualized as bands at specific distances from the top of the gel (the negative electrode end) on the basis of their size. The sizes of the nucleic acid samples can be estimated by comparing the distance with the molecular weight standard samples (also called DNA ladder).

Each fragment will be a band in the gel. A mixture of fragments of varying sizes appears as a long smear. If the fragments are too large, e.g. uncut genomic DNA, they will form a single large band at the top of the gel.

Circular plasmids move at different speeds depending on their conformation (nicked, linear, covalently bound, supercoiled, and circular single stranded). A supercoiled plasmid travels faster because it has less friction against agarose matrix than nicked or linear plasmid.

If you want to isolate a specific fragment you can cut the gel and extract the DNA.

 

 

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