Venus In Red, Plate 5
The liquid water that had been on the surface helped keep the tectonic plates nice and flexible, in a sense adding lubrication to the process of plate tectonics. But without the oceans, plate activity ground to a halt, locking the surface of Venus in place. Plate tectonics play a crucial role in regulating the amount of carbon dioxide in the atmosphere. Essentially, carbon binds to elements in dirt and rocks, and those dirt and rocks eventually get buried far beneath the surface over the course of millions of years as the plates rub up against each other and sink below each other.
Venus in Red, Plate 5
The Crazy Venus Plate is a sleek aluminum plate finely crafted from aircraft-grade alloys which allows the Venus a highly responsive performance. The unique fashion in which Crazy's PU Cushions sit further enhances the idea that these plates respond to your every action. The adjustable housing features a hex screw lock to secure your toe stop firmly.
H.C. WestermannA Close Call, 1965plate glass, spruce, pine, linoleum, fox head, paper decoupage, cloth, chicken feathers, putty, and brass plate15 x 14 7/8 x 9 1/2 in38.1 x 37.8 x 24.1 cm
In 2007, Venus Express discovered that a huge double atmospheric vortex exists at the south pole. Venus Express discovered, in 2011, that an ozone layer exists high in the atmosphere of Venus. On 29 January 2013, ESA scientists reported that the ionosphere of Venus streams outwards in a manner similar to "the ion tail seen streaming from a comet under similar conditions."
The stratigraphically oldest tessera terrains have consistently lower thermal emissivity than the surrounding basaltic plains measured by Venus Express and Magellan, indicating a different, possibly a more felsic, mineral assemblage. The mechanism to generate a large amount of felsic crust usually requires the presence of water ocean and plate tectonics, implying that habitable condition had existed on early Venus. However, the nature of tessera terrains is far from certain.
The principal difference between the two planets is the lack of evidence for plate tectonics on Venus, possibly because its crust is too strong to subduct without water to make it less viscous. This results in reduced heat loss from the planet, preventing it from cooling and providing a likely explanation for its lack of an internally generated magnetic field. Instead, Venus may lose its internal heat in periodic major resurfacing events.
The lack of an intrinsic magnetic field at Venus was surprising, given that it is similar to Earth in size and was expected to contain a dynamo at its core. A dynamo requires three things: a conducting liquid, rotation, and convection. The core is thought to be electrically conductive and, although its rotation is often thought to be too slow, simulations show it is adequate to produce a dynamo. This implies that the dynamo is missing because of a lack of convection in Venus's core. On Earth, convection occurs in the liquid outer layer of the core because the bottom of the liquid layer is much higher in temperature than the top. On Venus, a global resurfacing event may have shut down plate tectonics and led to a reduced heat flux through the crust. This insulating effect would cause the mantle temperature to increase, thereby reducing the heat flux out of the core. As a result, no internal geodynamo is available to drive a magnetic field. Instead, the heat from the core is reheating the crust.
Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.
Although the insertion of genes into plasmid vectors is amongst the most routinely performed procedures in molecular biology alongside PCR (polymerase chain reaction), the methods for the screening of successful gene insertion remains a tedious process that often involves running gel electrophoresis on restriction digestions or PCR reactions of many bacterial colonies to check for gene integration. To address this problem, the blue-white colorimetric screen was developed to allow on-plate screening of plasmid integration. This commonly used on-plate screening method employs an engineered lacZα protein with an internal multiple cloning site (MCS). In the presence of the chemical X-gal, β-galactosidase activity is detected via blue bacterial colonies. Insertion of PCR products within the MCS disrupts translation of lacZα preventing transformed bacteria from turning blue and thus, allowing for on-plate detection of successful gene integration. Complicating factors such as the spontaneous deletion of the lacZα gene during the cloning process and the insertion of genes that do not disrupt lacZα function lead to false-positive or false-negative screens, respectively, . In contrast to screening for the loss of lacZα function, fluorescent protein-based screening is based on a gain of fluorescence that can increase screening fidelity as the fluorescence property cannot be acquired spontaneously. Furthermore, exogenous chemical co-factors such as X-gal are not required and genes fused with fluorescent proteins are often the desired final products for further cell imaging studies.
Fluorescence screening of gene insertion on bacterial culture plates can be performed using the pCfvtx plasmid from Truong's cassette methodology because the successful insertion creates a C-terminal fusion of the gene with Venus (yellow fluorescent protein mutant). However, fluorescence screening is limited by the folding stability of the inserted gene as proper folding of the downstream fluorescent protein is directly related to the folding robustness of N-terminal proteins, . Furthermore, since gene cloning is performed in e. coli which may lack required chaperone proteins, the translated protein may mis-fold or aggregate in the host organism. These factors adversely affect the folding of the fluorescent protein, hindering successful screens using fluorescence. Here, we address the negative effects of protein folding on fluorescence screening with three alternative methods that utilize fluorescent proteins to screen for successful gene integration.
One method to enhance fluorescence screening is to promote the folding or enhanced expression of the gene insert by an N-terminal protein that is well-folded and expressed such as calmodulin (CaM). A poorly folded protein negatively affects folding of downstream proteins. However, a well-folded protein might facilitate increased protein expression levels by overcoming translation initiation. This results in the production of increased downstream fluorescent proteins allowing for improved fluorescence detection. In other studies, we have noticed that fusion protein containing CaM and Venus showed very bright fluorescence, suggesting that CaM is particularly well-folded. Thus, we constructed our plasmid expression vector with CaM situated N-terminal to the MCS1 (Fig. 1A). Testing of this plasmid vector for enhanced fluorescence screening was conducted by inserting the PCR fragment encoding the fluorescent protein hcRed (between MCS1 and MCS2) . Although it is not strictly necessary for the inserted gene to be fluorescent as gene insertion into our plasmid expression vector will induce Venus fluorescence for screening, hcRed was chosen because it also expresses fluorescence and can be used as a simple additional marker for gene insertion. When transformed colonies were plated on agar plates and incubated overnight at 37C, a substantial increase in Venus fluorescence intensity was observed relative to the standard insertion into the pCfvtx plasmid under a fluorescence plate reader (Fig. 2A). Enhanced fluorescence screening was also seen when other gene of varying length and folding efficiency was inserted into the N-terminally situated CaM plasmid expression vector (Fig. S1). To quantify the intensities of the Venus fluorescence, fluorescent colonies of both types were picked and grown overnight in LB broth. Bacterial culture growths were then adjusted to obtain approximately equal cell densities as measured by OD600 and fluorescence intensities were measured with a fluorometer. The expression vector with CaM showed 30.2x increased in fluorescence relative to the control whereas the standard insertion into the pCfvtx plasmid only showed a 3.2x increase over control (Fig. S2). To confirm that hcRed DNA fragments had been inserted, plasmids were extracted from fluorescent colonies and subjected to electrophoresis after restriction enzyme digestion. The effectiveness of this particular expression plasmid vector in discriminating bacterial colonies with successfully inserted gene fragments from unsuccessful colonies was conducted by a serial dilution of the hcRed insert to produce sub-optimal ligation reaction (Fig. S3).
Since the most N-terminal protein has the best opportunity for proper protein-folding and proper folding of our reporter fluorescent protein is imperative for efficient screening, another method to enhance on-plate fluorescence screening is creating gene inserts such that its integration will reconstitute a truncated N-terminal fluorescent protein. In this method, a reporter fluorescent protein was positioned N-terminal of the gene insert so that it could efficiently report the insertion of a desirable gene into the plasmid. Since fluorescence of the fluorescent protein depends on the proper formation of the β-barrel structure, , , fluorescence can be abolished by truncating the fluorescent protein at the C-terminus and disrupting this β-barrel structure. Then, by designing PCR fragments to include the last beta strand of the fluorescent protein, the inserted PCR product will complete the truncated fluorescent protein and restore fluorescence (Fig. 1B). It is important to note however, that there is a possibility that the downstream protein could be inserted with a frame shift after the rescuing last beta strand. This would result in the upstream fluorescent protein gaining the missing strand and expressing fluorescence however, the downstream protein would be out of frame and thus not expressed. Going back to our method, mRFP1 was chosen as the fluorescent protein for truncation as it is stable and matures quickly. Successive C-terminal truncation of mRFP1 showed that fluorescence was abolished after 10 amino acids were removed (Fig. S4, S5). Surprisingly, we found that mRFP1 fluorescence intensity increased after 7 amino acids were eliminated. In order to reduce cost and increase PCR efficiency, the ideal complementary strand to the truncated mRFP1 should be as short as possible while still allowing for fluorescence. Thus, PCR primers for Cerulean (cyan fluorescent protein mutant) were designed to methodically complete the truncated mRFP1. Again, Cerulean was chosen as it acts as an additional marker for gene insertion as well as providing a simple assay for detecting the presence of any frame shifts after the rescuing last beta strand as any frame shifts would abrogate Cerulean fluorescence. When these PCR fragments were inserted, we found that the addition of the amino acids RA to the truncated mRFP1 yielded the brightest fluorescent colonies (Fig. 2B). To confirm PCR fragments were successfully inserted, extracted plasmids from fluorescent colonies were sequenced. In addition, because the expression vector backbone contains a dual promoter system for expression in bacterial and mammalian cells, the final construct can be used directly in cell imaging studies. As a demonstration, the rescued truncated mRFP1 fused with Cerulean was directly transfected into three different cell lines (Fig. 3). 350c69d7ab