Optical Reporting Of Electrical Activity

 By restriction enzymes then amplified by polymerase chain reaction to make many to millions of copies of a single fragment.

- The most common method is with an agrobacterium. Since bacteria reproduce quickly, it’s easy to create the same

Extracellular recording electrodes were used to measure the compound action potentials (CAPs) in a cockroach leg nerve. CAPs are the summations of all present action potentials (APs) in the individual axons of the nerve. When an AP is conducted along an axon, sodium channels open and positively charged sodium ions enter the axon. Therefore the inside and the outside voltage changes. The voltage changes in the extracellular fluid were measured. A depolarisation of the axonal membrane causes a local negative charge in the extracellular fluid. The summation of all the voltage changes in the extracellular fluid at a specific position is measured by the recording electrodes.

Voltage-gated ion channels maintain the concentrations of different ions inside and outside of the neuron cell.

134). They are loops of DNA that are separate from the chromosomal DNA and can self-replicate in a cell, found mostly in bacteria (Brown, 2011; Addgene, 2015). Lederberg and William Hayes discovered that plasmids were being transferred from one cell to another, not the chromosomal DNA (Brown, 2011, p. 135). This discovery lead to plasmids being an essential tool for scientists. Scientists can engineer plasmids to have specific genes to introduce into new cells (Brown, 2011, p. 134). On a plasmid loop there will be an origin of replication (ORI) and a multiple cloning site (MCS) where the gene of interest is inserted (Bio-Rad, 2015). This region has specific restriction enzyme recognition sites, which are cut by the enzymes to open up the DNA where the new gene will be inserted (Jove Science Education Database, 2015). Most plasmids will also contain an antibiotic resistance gene allowing cell survival in environments containing antibiotics (Jove Science Education Database, 2015).

Spin the two tubes in a centrifuge for 5 minutes on opposite side of the centrifuge. The bacterium will collect at the bottom of the tube, so pour out the extraneous supinate. Then, add 250 microliters of buffer. The Ca2+ cation of the buffer neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to pass through the cell wall and enter the cells. Place both tubes on ice. Then add 10 microliters of water into one tube and 10 microliters of plasmid DNA into another tube labeling the one with DNA with a + and the one with water -, and place on ice for 10 minutes.

How do you insert the plasmid inside the bacteria? What process do you use? (1/2 pt)

This experiment’s results and mechanisms relates current medical disease which well known as autism spectrum disorder (ASD). Symptoms of ASD are difficulty of communication and interaction with others and inadequate eye contact. When GRIP proteins do not interact with AMPAR receptor, ASD were developed due to genetic mutation of the GRIP. GRIPs are located on the presynaptic and postsynaptic cell, but when diagnosing ASD disease, only postsynaptic cell were observed to detect ASD. The reason I am desiring to predict activity in presynaptic cell is to understand neuron activity. Knowing that allows to regulate protein and receptor and manipulate the neurons the way genetics want to cure ASD. If geneticist know how neurons

Figure 1. A Comparison of a CAP Recording of an Earthworm to an Intercellular Recording. The “taller” graph is a depiction of a microelectrode recording of an action potential inside a neuron. The highlighted graph is a depiction of an extracellular recording suction electrode on a giant earthworm. The dotted line represents the minimum voltage needed to depolarize an action potential. The results are obtained from a PowerLab Data Acquisition Unit and a LabChart computerized software. The data are recorded in units of milliseconds and millivolts.

This process uses enzymes to cut and insert piece of DNA into a plasmid vector. This vector is then transfected into cells. The different pieces of DNA that were being inserted into this plasmid were a control and three different mutants (an alpha-5-tailless mutant, full-length alpha-5, and full-length alpha-2). These pieces of DNA code for integrins. Once these pieces of DNA were inserted into the plasmid vector, the plasmid needed to be inserted into a cell where it would be able to replicate. In these tests, rat intestinal epithelial wild-type cells (cells that would be found in nature, not cells grown in the laboratory) were used. To insert the plasmid into these cells lipofectamine plus was used. Lipofectamine plus acts like a detergent and opens the membrane of the cell so the plasmid can enter. Also, the plasmids that were transfected into cells were all treated with G-418, an antibiotic. When the cells were plated and allowed to multiply, only those containing the plasmid with the antibiotic would live. This made it possible to know which cells actually received the plasmid and properly underwent mitosis, and to eliminate those cells that never took up the plasmid in the first place.

An action potential is a short electrical impulse generated at the axon hillock which travels the length of an axon. Its generation happens in three distinct stages, depolarisation, repolarisation and hyperpolarisation. When the threshold of excitation is reached, depolarisation starts, the threshold is between -55mV and -65mV in most neurons. When the neuron is stimulated voltage-activated Sodium (Na+) channels open, allowing Na+ ions to rush into the neuron. This reverses the polarity in the neuron towards its peak of +40mV. At this peak Na+ channels

A basic method in which we get specific genes integrated with another organism’s chromosome is as follows: Isolate the DNA from which selected gene is to be taken from and treat it with enzymes that will cut out that specific gene. These genes are then inserted into bacteria and grown into colonies being

This was done by using DNA ligation and E.Coli transformation. We looked at agar plates to analyze which one of E.coli cell strains took up the vector alone of the vector containing the gene of interest. Four agar plates were used in this laboratory which were labeled Ligation, pGEM- T Positive Control, Competent Cells Negative Control, and Competent Cells Positive Control. Reagents were used such as DNA Ligase Buffer, SOC Media, Ampicillin, IPTG, and X-Gal. Ligation was performed using 2X ligation buffer, DNA Mix, T4 DNA Ligase and sterile dH2O. Then the transformation of E. coli occurred in which our 4 LB agar plates were prepared with the corresponding amount of IPTG, X-Gal and ampicillin gene. The next step was pipetting the content into the corresponding plates and SOC media was added.The transformation tubes consent were placed on the agar plates using a spreader. The plates were incubated at 37 Celsius and then will be stored at 4 degrees Celsius.The ligation plate was prepared by adding 100 µL of IPTG, 50 µL of X-Gal and Ampicillin. In the ligation plate, the expected color to be see was blue. However, in our ligation plate, we were seeing both blue and white colonies. Blue cells indicate that the cells take up the vector alone in the presence of IPTG and X-Gal due to Beta- Galactosidase expression. IPTG or

The type of sensory neuron that would like respond to a green light would be photoreceptors.

Nerve cells generate electrical signals to transmit information. Neurons are not necessarily intrinsically great electrical conductors, however, they have evolved specialized mechanisms for propagating signals based on the flow of ions across their membranes.