http://2010.igem.org/wiki/index.php?title=Special:Contributions/Alalani&feed=atom&limit=50&target=Alalani&year=&month=2010.igem.org - User contributions [en]2024-03-28T23:52:01ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Georgia_State/WhyPichiaTeam:Georgia State/WhyPichia2010-10-28T03:55:31Z<p>Alalani: /* High production of foreign Protein while low levels of endogenous protein */</p>
<hr />
<div>{{Georgia_State/Header}}<br />
<br />
<br />
[[Image:Whypichia.png|500px|right]]<br />
<br />
<br />
=='''Pichia Vs Saccharomyces'''== <br />
<br />
<br />
'''Greater Cell Concentrations'''<br />
The preference of Pichia pastoris for respiratory growth is a key characteristic which allows it to be cultured at high cell densities (500 OD600 U ml31)[1]. This trait lends an advantage to Pichia over Saccharomyces cervisiae: Resultant ethanol from S. cervisiae fermentation accumulates which hinders culture growth and therefore protein production. High levels of cell growth in fermenter cultures are vital to foreign protein production since concentration of the secreted product in "extracellular medium is...proportional to the concentration of cells in the culture". [2] <br />
<br />
General notworthy differences inlcude the relatively stable ell membrane lipids of Pichia that form etter biocatalyst.(jiang, 2008). Pichia also has more compact and organized golgi bodies than Saccharomyces(Morawski, 2000). In contrast, there are many similarities between Pichia and Saccharomyces that greatly increase the benefits affiliated with using Pichia. Genetic similarity between the two organisms has enabled expression of similar genes and compatibility between vectors. Other similarities include identical pathways for TCA, pentose phosphate, glycolysis and amino acid synthesis(Fiaux, 2003) and (Baumann, 2010).<br />
<br />
=='''Easy and Inexpensive to Culture'''==<br />
<br />
<br />
The components of P. pastoris media (glycerol, methanol, salts, trace elements, and biotin) are relatively inexpensive and as such are well suited for large-scale heterologous protein production. The common protocol is to allow P. pastoris to accumulate biomass in a glycerol/carbon-source medium while foreign protein expression is repressed. Once glycerol stocks have been depleted, methanol is added to induce expression. Any undesirable protease activity can be easily averted by adjusting pH. Pichia's broad spectrum of optimal growth pH (3.0-7.0) provides ample wiggle room. A number of other techniques may be employed in minimizing proteolysis including lowering growth rate via methanol-level modulation, addition of protease inhibitors, addition of alternative protease substrates, and even simply lowering process temperature.<br />
<br />
<br />
== '''High production of foreign Protein while low levels of endogenous protein''' ==<br />
<br />
<br />
Extracellular expression of foreign proteins is favorable to intracellular production due to Pichia's extremely low levels of endogenous protein secretion, which makes it easier to isolate the protein of interest. Pichia vectors can also be cloned with foreign genes which align with a secretion signal (native to the protein, native to Pichia, or native to Saccharomyces), facilitating extracellular production. However, intracellular protein production is also high due to the efficiency--at carefully monitored methanol levels--of the AOX promoter. <br />
<br />
<br />
''' a. Large Upscale of Proteins''' <br />
<br />
Pichia pastoris is greatly used as a protein expression system because of several distinctive reasons. This methylotrophic yeast, P. pastoris, has a high growth rate and is able to grow on uncomplicated, low-cost medium. The presence of two strongly inducible promoters (AOX I & AOXII) allows P. pastoris to use methanol as its sole carbon and energy source. Large upscale (<130 g/l dry cell weight) can be maximized because of its preference for respiratory growth, which allows it to be cultured at high cell densities compared to the fermentative yeasts such as Saccharomyces cerevisiae. (Weidner et al). In turn, this allows for large yields of the recombinant proteins to be produced. The protein production of P. pastoris is largely extracellular because the amounts of endogenous proteins are limited. Secretion in this manner is in fact the first step of purification since the medium used to grow P. pastoris has no added proteins.<br />
<br />
== '''Pichia pastoris versus Esherichia coli''' ==<br />
<br />
<br />
The bacterium E. coli has been frequently chosen as a host to express recombinant, heterologous proteins. Although many successful experiments have used E. coli as their expression system, P. pastoris has become an increasingly popular alternative due to the shortcomings of E. coli. P. pastoris serves as a better host when attempting to express foreign genes because this yeast can perform post-translational modifications and fold proteins properly, tightly regulate transcription via its wide range of promotors, and be purified easily.<br />
<br />
a. Post-translational modifications<br />
<br />
Unlike P. pastoris, E. coli cannot perform higher eukaryotic post-translational modifications. As a result, proteins requiring these modifications often fold incorrectly when produced by E. coli. These disadvantages, which result from E. coli’s prokaryotic nature, limit the types of proteins that this system can express. Proteins that contain disulfide bonds or require post-translational modifications such as glycosylation, isomerization, or phosphorylation are not always properly expressed; they can be insoluble or improperly folded, which require additional solubilization and re-folding steps (Daly et al). Take for instance erythropoietin (EPO), a glycosylated protein. When EPO is expressed in E. coli, it is not glycosylated and becomes less resistant to unfolding compared to its natural, glycosylated form (Daly et al). Therefore, additional steps are needed in order to form a stable protein, which is time-consuming, costly, and has the tendency to produce low yields. <br />
<br />
A study done by Leuking et al. also supports the use of P. pastoris for protein expression. Vectors were created for both E. coli and P. pastoris using multiple cDNAs from a human fetal brain expression library. Out of the 29 DNA clones, all produced soluble proteins in P. pastoris, while E. coli was much less successful. In E. coli, only nine produced soluble proteins; 15 were detected as inclusion bodies; and five were not expressed at all. These differences are likely due to E. coli’s lack of eukaryotic abilities to perform post-translational modifications and properly fold proteins (Leuking et al).<br />
<br />
b. Powerful promoter systems<br />
<br />
P. pastoris’s wide range of promotors also aid in its use for foreign protein production. It has many strongly induced promotors, which allow sufficient expression of the genes of interest and therefore, a high concentration of proteins can be easily and inexpensively produced (Weidner et al). The alcohol oxidase genes, AOX1 and AOX2, are frequently used promotors because they are easily induced by methanol; because P. pastoris is methylotropic, meaning it can metabolize methanol as its carbon and energy source, adding methanol to the growth medium will induce these two promotors and then express the desired protein (Macauley-Patrick et al). High concentrations of protein can then be produced.<br />
A third advantage that P. pastoris has over E. coli is that proteins expressed by P. pastoris are easily purified (Weidner et al). Because P. pastoris secretes its recombinant protein in the growth medium and secretes low levels of endogeneous proteins, there is a better chance of obtaining high yields of uncontaminated proteins in comparison to E. coli (Weidner et al). In essence, there are fewer steps required to obtain the desired protein.<br />
<br />
<br />
Works Cited<br />
Daly, Rachel, and Milton T. W. Hearn. "Expression of Heterologous Proteins in Pichia pastoris: a Useful Experimental Tool in Protein Engineering and Production." PubMed.gov. PubMed, 26 Nov. 2004. Web. 25 Oct. 2010. <http://www.ncbi.nlm.nih.gov/pubmed/15565717>.<br />
Leuking, Angelika, Caterina Holz, Christine Gotthold, Hans Lehrach, and Dolores Cahill. “A System for Dual Protein Expression in Pichia pastoris and Escherichia coli.” PubMed.gov. PubMed, 15 Dec. 2003. Web. 25 Oct. 2010. <http://www.ncbi.nlm.nih.gov/pubmed/20186119>.<br />
Macauley-Patrick, Sue, Mariana L. Fazenda, Brian McNeil, and Linda M. "Heterologous Protein Production Using the Pichia pastoris Expression System." PubMed.gov. PubMed, 22 Mar. 2005. Web. 25 Oct. 2010. <http://www.ncbi.nlm.nih.gov/pubmed/15704221>.<br />
Weidner, Maria, Marcus Taupp, and Steven J. Hallam. "Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris." PubMed.gov. PubMed, 25 Feb. 2010. Web. 25 Oct. 2010. <http://www.ncbi.nlm.nih.gov/pubmed/20186119>.<br />
<br />
<br />
<br />
{{Georgia_State/Footer}}</div>Alalanihttp://2010.igem.org/Team:Georgia_State/WhyPichiaTeam:Georgia State/WhyPichia2010-10-28T03:53:34Z<p>Alalani: /* High production of foreign Protein while low levels of endogenous protein */</p>
<hr />
<div>{{Georgia_State/Header}}<br />
<br />
<br />
[[Image:Whypichia.png|500px|right]]<br />
<br />
<br />
=='''Pichia Vs Saccharomyces'''== <br />
<br />
<br />
'''Greater Cell Concentrations'''<br />
The preference of Pichia pastoris for respiratory growth is a key characteristic which allows it to be cultured at high cell densities (500 OD600 U ml31)[1]. This trait lends an advantage to Pichia over Saccharomyces cervisiae: Resultant ethanol from S. cervisiae fermentation accumulates which hinders culture growth and therefore protein production. High levels of cell growth in fermenter cultures are vital to foreign protein production since concentration of the secreted product in "extracellular medium is...proportional to the concentration of cells in the culture". [2] <br />
<br />
General notworthy differences inlcude the relatively stable ell membrane lipids of Pichia that form etter biocatalyst.(jiang, 2008). Pichia also has more compact and organized golgi bodies than Saccharomyces(Morawski, 2000). In contrast, there are many similarities between Pichia and Saccharomyces that greatly increase the benefits affiliated with using Pichia. Genetic similarity between the two organisms has enabled expression of similar genes and compatibility between vectors. Other similarities include identical pathways for TCA, pentose phosphate, glycolysis and amino acid synthesis(Fiaux, 2003) and (Baumann, 2010).<br />
<br />
=='''Easy and Inexpensive to Culture'''==<br />
<br />
<br />
The components of P. pastoris media (glycerol, methanol, salts, trace elements, and biotin) are relatively inexpensive and as such are well suited for large-scale heterologous protein production. The common protocol is to allow P. pastoris to accumulate biomass in a glycerol/carbon-source medium while foreign protein expression is repressed. Once glycerol stocks have been depleted, methanol is added to induce expression. Any undesirable protease activity can be easily averted by adjusting pH. Pichia's broad spectrum of optimal growth pH (3.0-7.0) provides ample wiggle room. A number of other techniques may be employed in minimizing proteolysis including lowering growth rate via methanol-level modulation, addition of protease inhibitors, addition of alternative protease substrates, and even simply lowering process temperature.<br />
<br />
<br />
== '''High production of foreign Protein while low levels of endogenous protein''' ==<br />
<br />
<br />
Extracellular expression of foreign proteins is favorable to intracellular production due to Pichia's extremely low levels of endogenous protein secretion, which makes it easier to isolate the protein of interest. Pichia vectors can also be cloned with foreign genes which align with a secretion signal (native to the protein, native to Pichia, or native to Saccharomyces), facilitating extracellular production. However, intracellular protein production is also high due to the efficiency--at carefully monitored methanol levels--of the AOX promoter. <br />
<br />
<br />
a. Large Upscale of Proteins <br />
Pichia pastoris is greatly used as a protein expression system because of several distinctive reasons. This methylotrophic yeast, P. pastoris, has a high growth rate and is able to grow on uncomplicated, low-cost medium. The presence of two strongly inducible promoters (AOX I & AOXII) allows P. pastoris to use methanol as its sole carbon and energy source. Large upscale (<130 g/l dry cell weight) can be maximized because of its preference for respiratory growth, which allows it to be cultured at high cell densities compared to the fermentative yeasts such as Saccharomyces cerevisiae. (Weidner et al). In turn, this allows for large yields of the recombinant proteins to be produced. The protein production of P. pastoris is largely extracellular because the amounts of endogenous proteins are limited. Secretion in this manner is in fact the first step of purification since the medium used to grow P. pastoris has no added proteins.<br />
<br />
== '''Pichia pastoris versus Esherichia coli''' ==<br />
<br />
<br />
The bacterium E. coli has been frequently chosen as a host to express recombinant, heterologous proteins. Although many successful experiments have used E. coli as their expression system, P. pastoris has become an increasingly popular alternative due to the shortcomings of E. coli. P. pastoris serves as a better host when attempting to express foreign genes because this yeast can perform post-translational modifications and fold proteins properly, tightly regulate transcription via its wide range of promotors, and be purified easily.<br />
<br />
a. Post-translational modifications<br />
<br />
Unlike P. pastoris, E. coli cannot perform higher eukaryotic post-translational modifications. As a result, proteins requiring these modifications often fold incorrectly when produced by E. coli. These disadvantages, which result from E. coli’s prokaryotic nature, limit the types of proteins that this system can express. Proteins that contain disulfide bonds or require post-translational modifications such as glycosylation, isomerization, or phosphorylation are not always properly expressed; they can be insoluble or improperly folded, which require additional solubilization and re-folding steps (Daly et al). Take for instance erythropoietin (EPO), a glycosylated protein. When EPO is expressed in E. coli, it is not glycosylated and becomes less resistant to unfolding compared to its natural, glycosylated form (Daly et al). Therefore, additional steps are needed in order to form a stable protein, which is time-consuming, costly, and has the tendency to produce low yields. <br />
<br />
A study done by Leuking et al. also supports the use of P. pastoris for protein expression. Vectors were created for both E. coli and P. pastoris using multiple cDNAs from a human fetal brain expression library. Out of the 29 DNA clones, all produced soluble proteins in P. pastoris, while E. coli was much less successful. In E. coli, only nine produced soluble proteins; 15 were detected as inclusion bodies; and five were not expressed at all. These differences are likely due to E. coli’s lack of eukaryotic abilities to perform post-translational modifications and properly fold proteins (Leuking et al).<br />
<br />
b. Powerful promoter systems<br />
<br />
P. pastoris’s wide range of promotors also aid in its use for foreign protein production. It has many strongly induced promotors, which allow sufficient expression of the genes of interest and therefore, a high concentration of proteins can be easily and inexpensively produced (Weidner et al). The alcohol oxidase genes, AOX1 and AOX2, are frequently used promotors because they are easily induced by methanol; because P. pastoris is methylotropic, meaning it can metabolize methanol as its carbon and energy source, adding methanol to the growth medium will induce these two promotors and then express the desired protein (Macauley-Patrick et al). High concentrations of protein can then be produced.<br />
A third advantage that P. pastoris has over E. coli is that proteins expressed by P. pastoris are easily purified (Weidner et al). Because P. pastoris secretes its recombinant protein in the growth medium and secretes low levels of endogeneous proteins, there is a better chance of obtaining high yields of uncontaminated proteins in comparison to E. coli (Weidner et al). In essence, there are fewer steps required to obtain the desired protein.<br />
<br />
<br />
Works Cited<br />
Daly, Rachel, and Milton T. W. Hearn. "Expression of Heterologous Proteins in Pichia pastoris: a Useful Experimental Tool in Protein Engineering and Production." PubMed.gov. PubMed, 26 Nov. 2004. Web. 25 Oct. 2010. <http://www.ncbi.nlm.nih.gov/pubmed/15565717>.<br />
Leuking, Angelika, Caterina Holz, Christine Gotthold, Hans Lehrach, and Dolores Cahill. “A System for Dual Protein Expression in Pichia pastoris and Escherichia coli.” PubMed.gov. PubMed, 15 Dec. 2003. Web. 25 Oct. 2010. <http://www.ncbi.nlm.nih.gov/pubmed/20186119>.<br />
Macauley-Patrick, Sue, Mariana L. Fazenda, Brian McNeil, and Linda M. "Heterologous Protein Production Using the Pichia pastoris Expression System." PubMed.gov. PubMed, 22 Mar. 2005. Web. 25 Oct. 2010. <http://www.ncbi.nlm.nih.gov/pubmed/15704221>.<br />
Weidner, Maria, Marcus Taupp, and Steven J. Hallam. "Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris." PubMed.gov. PubMed, 25 Feb. 2010. Web. 25 Oct. 2010. <http://www.ncbi.nlm.nih.gov/pubmed/20186119>.<br />
<br />
<br />
<br />
{{Georgia_State/Footer}}</div>Alalanihttp://2010.igem.org/Team:Georgia_State/ProtocolsTeam:Georgia State/Protocols2010-10-28T03:38:31Z<p>Alalani: /* Preparation of Electrocompetent cells */</p>
<hr />
<div><br />
{{Georgia_State/Header}}<br />
<br />
<br />
== '''Preparation of Electrocompetent cells''' ==<br />
<br />
1. Inoculate 500ml of L-broth with 1/100 volume of a fresh overnight E.coli culture<br />
<br />
2. Grow the cells at 37°C on shaker to an OD600.(dilute culture if desired OD is exceeded.<br />
<br />
a. Best results are obtained with cells harvested at early to mid log phase so the desired OD may vary based on specific strain growth conditions<br />
<br />
3. Chill cells on Ice for approximately 20 minutes. Keep cells on Ice for all subsequent steps in procedure and pre-chill all tubes before adding cells. If possible, centrifuge at 4°C.<br />
<br />
4. Transfer cells to chilled 50 ml Falcon tubes and centrifuge at 4000x g for 15 minutes<br />
<br />
5. Pour off and discard supernatant. Resuspend pellet in 50ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 min. Pour off and discard supernatant.<br />
<br />
6. Resuspend pellet in 25 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 min. Pour off and discard supernatant.<br />
<br />
7. Resuspend Pellet in 20 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 min. Pour off and discard supernatant.<br />
<br />
8. Resuspend cell pellet in a final volume of 2ml of ice-cold 10% glycerol. Cell concentration should be about 1-3 x 1010 cells/ml<br />
<br />
9. Make aliquots of 100µl and store at -80°C.<br />
<br />
== '''Preparation of Heat Shock Competent Cells''' ==<br />
<br />
Preparation of Seed Stock<br />
1. Streak TOP 10 cells on an SOB plate and grow for single colonies at room temperature<br />
2. Pick single colonies into 2mL of SOB medium and shake overnight at room temperature<br />
3. Add 15% glycerol <br />
4. Aliquot into 1mL samples<br />
5. Place in -80°C<br />
Preparing competent cells <br />
1. Prechill 2mL centrifuge tubes<br />
2. Inoculate 250mL of SOB medium with 1mL vial of seed stock and grow at 20°C to an OD600nm of 0.3<br />
3. Aim for a lower OD not higher if possible for 16 hours at room temperature should work<br />
4. Centrifuge at 3000g at 4°C for 10 minutes<br />
5. Pellets should be resuspended in 80mL of ice cold ccMB80 buffer<br />
6. Incubate on ice for 20 minutes<br />
7. Centrifuge again at 4°C and resuspend in 10mL of ice cold ccMB80 buffer<br />
8. Test the OD of a mixture of 200uL of SOC and 50uL of the resuspended cells<br />
9. Add chilled ccMB80 to yield a final OD of 1.0 – 1.5<br />
10. Incubate on ice for twenty minutes<br />
11. Aliquot into chilled 2mL chilled centrifuge tubes<br />
12. Store at – 80°C<br />
<br />
<br />
== '''Transformation of DNA using Electroporation''' ==<br />
<br />
1. Thaw DNA and cells<br />
2. Add 40uL of cells in a fresh microcentrifuge tube along with 1uL of DNA<br />
3. After gently mixing transfer the contents of the microcentrifuge tube into an electroporation cuvette<br />
4. Have the micropulsor set to Eco1<br />
5. Hit the pulse button and hold it until you hear it beep<br />
6. Immediately add 1mL of SOC broth to the electroporated cells<br />
7. Transfer the contents of the cuvette to a micro centrifuge tube and inoculate the tubes at 37°C for an hour<br />
8. Plate 20uL and 100uL on selective media plates<br />
9. Also plate 100uL on regular media plates as a control to test the viability of the shocked cells<br />
10. Inoculate these plates overnight for 14-16hrs at 37°C <br />
<br />
<br />
<br />
== '''Transformation Using Heat Shock''' ==<br />
<br />
<br />
1. Thaw the cells and DNA on ice<br />
2. In a microcentrifuge tube at 50uL of competent cells with 2uL of DNA<br />
3. Let it sit on ice for 30minutes<br />
4. Heat shock at 42°C for 60seconds<br />
5. Let it sit on ice for 5minutes<br />
6. Add 200uL of SOC broth<br />
7. Incubate at 37°C for 2hours<br />
8. Plate 20uL and 100uL on Selective media<br />
9. Plate 100uL on normal media as a control to test viability of the heat shock cells<br />
<br />
<br />
{{Georgia_State/Footer}}</div>Alalanihttp://2010.igem.org/Team:Georgia_State/ProtocolsTeam:Georgia State/Protocols2010-10-28T03:36:40Z<p>Alalani: New page: {{Georgia_State/Header}} == '''Preparation of Electrocompetent cells''' == 1. Inoculate 500ml of L-broth with 1/100 volume of a fresh overnight E.coli culture 2. Grow the cells at 37...</p>
<hr />
<div><br />
{{Georgia_State/Header}}<br />
<br />
<br />
== '''Preparation of Electrocompetent cells''' ==<br />
<br />
1. Inoculate 500ml of L-broth with 1/100 volume of a fresh overnight E.coli culture<br />
2. Grow the cells at 37°C on shaker to an OD600.(dilute culture if desired OD is exceeded.)<br />
a. Best results are obtained with cells harvested at early to mid log phase so the desired OD may vary based on specific strain growth conditions<br />
3. Chill cells on Ice for approximately 20 minutes. Keep cells on Ice for all subsequent steps in procedure and pre-chill all tubes before adding cells. If possible, centrifuge at 4°C.<br />
4. Transfer cells to chilled 50 ml Falcon tubes and centrifuge at 4000x g for 15 minutes<br />
5. Pour off and discard supernatant. Resuspend pellet in 50ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 min. Pour off and discard supernatant.<br />
6. Resuspend pellet in 25 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 min. Pour off and discard supernatant.<br />
7. Resuspend Pellet in 20 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 min. Pour off and discard supernatant.<br />
8. Resuspend cell pellet in a final volume of 2ml of ice-cold 10% glycerol. Cell concentration should be about 1-3 x 1010 cells/ml<br />
9. Make aliquots of 100µl and store at -80°C.<br />
<br />
<br />
<br />
== '''Preparation of Heat Shock Competent Cells''' ==<br />
<br />
Preparation of Seed Stock<br />
1. Streak TOP 10 cells on an SOB plate and grow for single colonies at room temperature<br />
2. Pick single colonies into 2mL of SOB medium and shake overnight at room temperature<br />
3. Add 15% glycerol <br />
4. Aliquot into 1mL samples<br />
5. Place in -80°C<br />
Preparing competent cells <br />
1. Prechill 2mL centrifuge tubes<br />
2. Inoculate 250mL of SOB medium with 1mL vial of seed stock and grow at 20°C to an OD600nm of 0.3<br />
3. Aim for a lower OD not higher if possible for 16 hours at room temperature should work<br />
4. Centrifuge at 3000g at 4°C for 10 minutes<br />
5. Pellets should be resuspended in 80mL of ice cold ccMB80 buffer<br />
6. Incubate on ice for 20 minutes<br />
7. Centrifuge again at 4°C and resuspend in 10mL of ice cold ccMB80 buffer<br />
8. Test the OD of a mixture of 200uL of SOC and 50uL of the resuspended cells<br />
9. Add chilled ccMB80 to yield a final OD of 1.0 – 1.5<br />
10. Incubate on ice for twenty minutes<br />
11. Aliquot into chilled 2mL chilled centrifuge tubes<br />
12. Store at – 80°C<br />
<br />
<br />
== '''Transformation of DNA using Electroporation''' ==<br />
<br />
1. Thaw DNA and cells<br />
2. Add 40uL of cells in a fresh microcentrifuge tube along with 1uL of DNA<br />
3. After gently mixing transfer the contents of the microcentrifuge tube into an electroporation cuvette<br />
4. Have the micropulsor set to Eco1<br />
5. Hit the pulse button and hold it until you hear it beep<br />
6. Immediately add 1mL of SOC broth to the electroporated cells<br />
7. Transfer the contents of the cuvette to a micro centrifuge tube and inoculate the tubes at 37°C for an hour<br />
8. Plate 20uL and 100uL on selective media plates<br />
9. Also plate 100uL on regular media plates as a control to test the viability of the shocked cells<br />
10. Inoculate these plates overnight for 14-16hrs at 37°C <br />
<br />
<br />
<br />
== '''Transformation Using Heat Shock''' ==<br />
<br />
<br />
1. Thaw the cells and DNA on ice<br />
2. In a microcentrifuge tube at 50uL of competent cells with 2uL of DNA<br />
3. Let it sit on ice for 30minutes<br />
4. Heat shock at 42°C for 60seconds<br />
5. Let it sit on ice for 5minutes<br />
6. Add 200uL of SOC broth<br />
7. Incubate at 37°C for 2hours<br />
8. Plate 20uL and 100uL on Selective media<br />
9. Plate 100uL on normal media as a control to test viability of the heat shock cells<br />
<br />
<br />
{{Georgia_State/Footer}}</div>Alalanihttp://2010.igem.org/Team:Georgia_State/ProducingtheantigenTeam:Georgia State/Producingtheantigen2010-10-28T03:17:47Z<p>Alalani: </p>
<hr />
<div>{{Georgia_State/Header}}<br />
<br />
<br />
<br />
== '''Designing the Antigen''' ==<br />
<br />
<br />
<br />
In order to design an antigen we compared previously isolated and sequenced Influenza A H1N1 strains. We were able to identify a conserved region that comprised of the Hemagglutinin globular head which is formed by a disulfide bond between two cysteins. This region of the virus has been shown to produce an immune response. This is important since we plan on being able to use this as a potential vaccine. In order to identify the location of the cysteins within the sequence, we converted the nucleotide sequence to the amino acid sequence. The cysteins were identified, and our part was designed to include this region. The next step was to design this part to fit the registry standard. Two unwanted restriction sites were identified. In order to eliminate these sites, we did a nucleotide swap while maintaining the same amino acid sequence. Following the above steps, the part has been designed to be compatible with all assembly standards.<br />
<br />
<br />
{{Georgia_State/Footer}}</div>Alalanihttp://2010.igem.org/Team:Georgia_State/ProducingtheantigenTeam:Georgia State/Producingtheantigen2010-10-28T03:17:30Z<p>Alalani: </p>
<hr />
<div>{{Georgia_State/Header}}<br />
<br />
<br />
== '''Designing the Antigen''' ==<br />
<br />
<br />
In order to design an antigen we compared previously isolated and sequenced Influenza A H1N1 strains. We were able to identify a conserved region that comprised of the Hemagglutinin globular head which is formed by a disulfide bond between two cysteins. This region of the virus has been shown to produce an immune response. This is important since we plan on being able to use this as a potential vaccine. In order to identify the location of the cysteins within the sequence, we converted the nucleotide sequence to the amino acid sequence. The cysteins were identified, and our part was designed to include this region. The next step was to design this part to fit the registry standard. Two unwanted restriction sites were identified. In order to eliminate these sites, we did a nucleotide swap while maintaining the same amino acid sequence. Following the above steps, the part has been designed to be compatible with all assembly standards.<br />
<br />
<br />
{{Georgia_State/Footer}}</div>Alalanihttp://2010.igem.org/Team:Georgia_State/ProducingtheantigenTeam:Georgia State/Producingtheantigen2010-10-28T03:16:51Z<p>Alalani: </p>
<hr />
<div>{{Georgia_State/Header}}<br />
<br />
== '''Designing the Antigen''' ==<br />
<br />
<br />
In order to design an antigen we compared previously isolated and sequenced Influenza A H1N1 strains. We were able to identify a conserved region that comprised of the Hemagglutinin globular head which is formed by a disulfide bond between two cysteins. This region of the virus has been shown to produce an immune response. This is important since we plan on being able to use this as a potential vaccine. In order to identify the location of the cysteins within the sequence, we converted the nucleotide sequence to the amino acid sequence. The cysteins were identified, and our part was designed to include this region. The next step was to design this part to fit the registry standard. Two unwanted restriction sites were identified. In order to eliminate these sites, we did a nucleotide swap while maintaining the same amino acid sequence. Following the above steps, the part has been designed to be compatible with all assembly standards.</div>Alalanihttp://2010.igem.org/Team:Georgia_State/ProducingtheantigenTeam:Georgia State/Producingtheantigen2010-10-28T03:16:09Z<p>Alalani: </p>
<hr />
<div><br />
<br />
== '''Designing the Antigen''' ==<br />
<br />
<br />
In order to design an antigen we compared previously isolated and sequenced Influenza A H1N1 strains. We were able to identify a conserved region that comprised of the Hemagglutinin globular head which is formed by a disulfide bond between two cysteins. This region of the virus has been shown to produce an immune response. This is important since we plan on being able to use this as a potential vaccine. In order to identify the location of the cysteins within the sequence, we converted the nucleotide sequence to the amino acid sequence. The cysteins were identified, and our part was designed to include this region. The next step was to design this part to fit the registry standard. Two unwanted restriction sites were identified. In order to eliminate these sites, we did a nucleotide swap while maintaining the same amino acid sequence. Following the above steps, the part has been designed to be compatible with all assembly standards.</div>Alalanihttp://2010.igem.org/Team:Georgia_State/ProducingtheantigenTeam:Georgia State/Producingtheantigen2010-10-28T03:15:49Z<p>Alalani: New page: == '''Designing the Antigen''' == In order to design an antigen we compared previously isolated and sequenced Influenza A H1N1 strains. We were able to identify a conserved region tha...</p>
<hr />
<div><br />
<br />
== '''Designing the Antigen''' ==<br />
<br />
<br />
<br />
In order to design an antigen we compared previously isolated and sequenced Influenza A H1N1 strains. We were able to identify a conserved region that comprised of the Hemagglutinin globular head which is formed by a disulfide bond between two cysteins. This region of the virus has been shown to produce an immune response. This is important since we plan on being able to use this as a potential vaccine. In order to identify the location of the cysteins within the sequence, we converted the nucleotide sequence to the amino acid sequence. The cysteins were identified, and our part was designed to include this region. The next step was to design this part to fit the registry standard. Two unwanted restriction sites were identified. In order to eliminate these sites, we did a nucleotide swap while maintaining the same amino acid sequence. Following the above steps, the part has been designed to be compatible with all assembly standards.</div>Alalanihttp://2010.igem.org/Team:Georgia_State/OursystemTeam:Georgia State/Oursystem2010-10-28T01:53:57Z<p>Alalani: </p>
<hr />
<div>{{Georgia_State/Header}}<br />
<br />
<br />
<br />
<br />
<br />
== H1N1 VACCINE ==<br />
<br />
<br />
<br />
'''Background''':<br />
In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A <br />
virus to the 6 phase alert. This is the highest alert level and it indicates widespread<br />
community transmission of the virus in over two continents. The H1N1 Influenza A virus<br />
is a quadruple reassortment of two swine strains, one human strain, and one avian strain.<br />
More than 214 countries and territories have reported laboratory – confirmed cases of<br />
pandemic H1N1 Influenza A. The US Centers for Disease Control and Prevention reported<br />
between April 2009 and April 2010, approximately 61million cases of pandemic H1N1 occurred,<br />
including 274,000 hospitalizations and 12,470 deaths. <br />
<br />
'''Problem:''' <br />
Efficient and timely vaccine production was a challenge for the scientific<br />
community during the pandemic outbreak of H1N1. Another problem with the <br />
Influenza viruses is the high variability and mutations that they present<br />
with each year. This makes vaccine production time difficult and time consuming.<br />
<br />
'''The Pichia System:'''<br />
The Pichia system that we plan to establish has the capability of overcoming this<br />
problem mentioned above. We have designed a standard Pichia system that can be<br />
used for the production of various different vaccines.The system involves an<br />
interchangeable antigen part that is designed based on the variant of the virus<br />
antigen that needs to be produced or any other antigen that needs to be produced. <br />
To illustrate this we present the globular head region of the H1N1 Influenza A virus.<br />
<br />
[[Image:H1N1.png|Center|atl=Alt text|H1N1 INFLUENZA A VIRUS]]<br />
<br />
<br />
{{Georgia_State/Footer}}</div>Alalanihttp://2010.igem.org/Team:Georgia_State/OursystemTeam:Georgia State/Oursystem2010-10-28T01:51:49Z<p>Alalani: </p>
<hr />
<div>{{Georgia_State/Header}}<br />
<br />
<br />
<br />
== H1N1 VACCINE ==<br />
<br />
<br />
<br />
'''Background''':<br />
In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A <br />
virus to the 6 phase alert. This is the highest alert level and it indicates widespread<br />
community transmission of the virus in over two continents. The H1N1 Influenza A virus<br />
is a quadruple reassortment of two swine strains, one human strain, and one avian strain.<br />
More than 214 countries and territories have reported laboratory – confirmed cases of<br />
pandemic H1N1 Influenza A. The US Centers for Disease Control and Prevention reported<br />
between April 2009 and April 2010, approximately 61million cases of pandemic H1N1 occurred,<br />
including 274,000 hospitalizations and 12,470 deaths. <br />
<br />
'''Problem:''' <br />
Efficient and timely vaccine production was a challenge for the scientific<br />
community during the pandemic outbreak of H1N1. Another problem with the <br />
Influenza viruses is the high variability and mutations that they present<br />
with each year. This makes vaccine production time difficult and time consuming.<br />
<br />
'''The Pichia System:'''<br />
The Pichia system that we plan to establish has the capability of overcoming this<br />
problem mentioned above. We have designed a standard Pichia system that can be<br />
used for the production of various different vaccines.The system involves an<br />
interchangeable antigen part that is designed based on the variant of the virus<br />
antigen that needs to be produced or any other antigen that needs to be produced. <br />
To illustrate this we present the globular head region of the H1N1 Influenza A virus.<br />
<br />
[[Image:H1N1.png|Center|atl=Alt text|H1N1 INFLUENZA A VIRUS]]<br />
<br />
<br />
{{Georgia_State/Footer}}</div>Alalanihttp://2010.igem.org/Team:Georgia_State/OursystemTeam:Georgia State/Oursystem2010-10-28T01:34:51Z<p>Alalani: </p>
<hr />
<div><br />
== H1N1 VACCINE ==<br />
<br />
<br />
<br />
'''Background''':<br />
In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A <br />
virus to the 6 phase alert. This is the highest alert level and it indicates widespread<br />
community transmission of the virus in over two continents. The H1N1 Influenza A virus<br />
is a quadruple reassortment of two swine strains, one human strain, and one avian strain.<br />
More than 214 countries and territories have reported laboratory – confirmed cases of<br />
pandemic H1N1 Influenza A. The US Centers for Disease Control and Prevention reported<br />
between April 2009 and April 2010, approximately 61million cases of pandemic H1N1 occurred,<br />
including 274,000 hospitalizations and 12,470 deaths. <br />
<br />
'''Problem:''' <br />
Efficient and timely vaccine production was a challenge for the scientific<br />
community during the pandemic outbreak of H1N1. Another problem with the <br />
Influenza viruses is the high variability and mutations that they present<br />
with each year. This makes vaccine production time difficult and time consuming.<br />
<br />
'''The Pichia System:'''<br />
The Pichia system that we plan to establish has the capability of overcoming this<br />
problem mentioned above. We have designed a standard Pichia system that can be<br />
used for the production of various different vaccines.The system involves an<br />
interchangeable antigen part that is designed based on the variant of the virus<br />
antigen that needs to be produced or any other antigen that needs to be produced. <br />
To illustrate this we present the globular head region of the H1N1 Influenza A virus.<br />
<br />
[[Image:H1N1.png|Center|atl=Alt text|H1N1 INFLUENZA A VIRUS]]</div>Alalanihttp://2010.igem.org/Team:Georgia_State/OursystemTeam:Georgia State/Oursystem2010-10-28T01:31:10Z<p>Alalani: </p>
<hr />
<div>'''<br />
== H1N1 VACCINE<br />
=='''<br />
<br />
'''Background''':<br />
In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A <br />
virus to the 6 phase alert. This is the highest alert level and it indicates widespread<br />
community transmission of the virus in over two continents. The H1N1 Influenza A virus<br />
is a quadruple reassortment of two swine strains, one human strain, and one avian strain.<br />
More than 214 countries and territories have reported laboratory – confirmed cases of<br />
pandemic H1N1 Influenza A. The US Centers for Disease Control and Prevention reported<br />
between April 2009 and April 2010, approximately 61million cases of pandemic H1N1 occurred,<br />
including 274,000 hospitalizations and 12,470 deaths. <br />
<br />
'''Problem:''' <br />
Efficient and timely vaccine production was a challenge for the scientific<br />
community during the pandemic outbreak of H1N1. Another problem with the <br />
Influenza viruses is the high variability and mutations that they present<br />
with each year. This makes vaccine production time difficult and time consuming.<br />
<br />
'''The Pichia System:'''<br />
The Pichia system that we plan to establish has the capability of overcoming this<br />
problem mentioned above. We have designed a standard Pichia system that can be<br />
used for the production of various different vaccines.The system involves an<br />
interchangeable antigen part that is designed based on the variant of the virus<br />
antigen that needs to be produced or any other antigen that needs to be produced. <br />
To illustrate this we present the globular head region of the H1N1 Influenza A virus.<br />
<br />
[[Image:H1N1.png|Center|atl=Alt text|H1N1 INFLUENZA A VIRUS]]</div>Alalanihttp://2010.igem.org/Team:Georgia_State/OursystemTeam:Georgia State/Oursystem2010-10-28T01:28:21Z<p>Alalani: </p>
<hr />
<div><br />
<br />
<br />
<br />
'''Background''':<br />
In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A <br />
virus to the 6 phase alert. This is the highest alert level and it indicates widespread<br />
community transmission of the virus in over two continents. The H1N1 Influenza A virus<br />
is a quadruple reassortment of two swine strains, one human strain, and one avian strain.<br />
More than 214 countries and territories have reported laboratory – confirmed cases of<br />
pandemic H1N1 Influenza A. The US Centers for Disease Control and Prevention reported<br />
between April 2009 and April 2010, approximately 61million cases of pandemic H1N1 occurred,<br />
including 274,000 hospitalizations and 12,470 deaths. <br />
<br />
'''Problem:''' <br />
Efficient and timely vaccine production was a challenge for the scientific<br />
community during the pandemic outbreak of H1N1. Another problem with the <br />
Influenza viruses is the high variability and mutations that they present<br />
with each year. This makes vaccine production time difficult and time consuming.<br />
<br />
'''The Pichia System:'''<br />
The Pichia system that we plan to establish has the capability of overcoming this<br />
problem mentioned above. We have designed a standard Pichia system that can be<br />
used for the production of various different vaccines.The system involves an<br />
interchangeable antigen part that is designed based on the variant of the virus<br />
antigen that needs to be produced or any other antigen that needs to be produced. <br />
To illustrate this we present the globular head region of the H1N1 Influenza A virus.<br />
<br />
[[Image:H1N1.png|Center|atl=Alt text|H1N1 INFLUENZA A VIRUS]]</div>Alalanihttp://2010.igem.org/Team:Georgia_State/OursystemTeam:Georgia State/Oursystem2010-10-28T01:26:57Z<p>Alalani: </p>
<hr />
<div>[[Image:H1N1.png|bottom|atl=Alt text|H1N1 INFLUENZA A VIRUS]]<br />
<br />
----<br />
'''Background''':<br />
In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A <br />
virus to the 6 phase alert. This is the highest alert level and it indicates widespread<br />
community transmission of the virus in over two continents. The H1N1 Influenza A virus<br />
is a quadruple reassortment of two swine strains, one human strain, and one avian strain.<br />
More than 214 countries and territories have reported laboratory – confirmed cases of<br />
pandemic H1N1 Influenza A. The US Centers for Disease Control and Prevention reported<br />
between April 2009 and April 2010, approximately 61million cases of pandemic H1N1 occurred,<br />
including 274,000 hospitalizations and 12,470 deaths. <br />
<br />
'''Problem:''' <br />
Efficient and timely vaccine production was a challenge for the scientific<br />
community during the pandemic outbreak of H1N1. Another problem with the <br />
Influenza viruses is the high variability and mutations that they present<br />
with each year. This makes vaccine production time difficult and time consuming.<br />
<br />
'''The Pichia System:'''<br />
The Pichia system that we plan to establish has the capability of overcoming this<br />
problem mentioned above. We have designed a standard Pichia system that can be<br />
used for the production of various different vaccines.The system involves an<br />
interchangeable antigen part that is designed based on the variant of the virus<br />
antigen that needs to be produced or any other antigen that needs to be produced. <br />
To illustrate this we present the globular head region of the H1N1 Influenza A virus.</div>Alalanihttp://2010.igem.org/Team:Georgia_State/OursystemTeam:Georgia State/Oursystem2010-10-28T01:25:14Z<p>Alalani: </p>
<hr />
<div>[[Image:H1N1.png|right|atl=Alt text|H1N1 INFLUENZA A VIRUS]]<br />
<br />
----<br />
'''Background''':<br />
In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A <br />
virus to the 6 phase alert. This is the highest alert level and it indicates widespread<br />
community transmission of the virus in over two continents. The H1N1 Influenza A virus<br />
is a quadruple reassortment of two swine strains, one human strain, and one avian strain.<br />
More than 214 countries and territories have reported laboratory – confirmed cases of<br />
pandemic H1N1 Influenza A. The US Centers for Disease Control and Prevention reported<br />
between April 2009 and April 2010, approximately 61million cases of pandemic H1N1 occurred,<br />
including 274,000 hospitalizations and 12,470 deaths. <br />
<br />
'''Problem:''' <br />
Efficient and timely vaccine production was a challenge for the scientific<br />
community during the pandemic outbreak of H1N1. Another problem with the <br />
Influenza viruses is the high variability and mutations that they present<br />
with each year. This makes vaccine production time difficult and time consuming.<br />
<br />
'''The Pichia System:'''<br />
The Pichia system that we plan to establish has the capability of overcoming this<br />
problem mentioned above. We have designed a standard Pichia system that can be<br />
used for the production of various different vaccines.The system involves an<br />
interchangeable antigen part that is designed based on the variant of the virus<br />
antigen that needs to be produced or any other antigen that needs to be produced. <br />
To illustrate this we present the globular head region of the H1N1 Influenza A virus.</div>Alalanihttp://2010.igem.org/File:H1N1.pngFile:H1N1.png2010-10-28T01:21:09Z<p>Alalani: </p>
<hr />
<div></div>Alalanihttp://2010.igem.org/Team:Georgia_State/OursystemTeam:Georgia State/Oursystem2010-10-28T01:17:03Z<p>Alalani: </p>
<hr />
<div><br />
----<br />
'''Background''':<br />
In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A <br />
virus to the 6 phase alert. This is the highest alert level and it indicates widespread<br />
community transmission of the virus in over two continents. The H1N1 Influenza A virus<br />
is a quadruple reassortment of two swine strains, one human strain, and one avian strain.<br />
More than 214 countries and territories have reported laboratory – confirmed cases of<br />
pandemic H1N1 Influenza A. The US Centers for Disease Control and Prevention reported<br />
between April 2009 and April 2010, approximately 61million cases of pandemic H1N1 occurred,<br />
including 274,000 hospitalizations and 12,470 deaths. <br />
<br />
'''Problem:''' <br />
Efficient and timely vaccine production was a challenge for the scientific<br />
community during the pandemic outbreak of H1N1. Another problem with the <br />
Influenza viruses is the high variability and mutations that they present<br />
with each year. This makes vaccine production time difficult and time consuming.<br />
<br />
'''The Pichia System:'''<br />
The Pichia system that we plan to establish has the capability of overcoming this<br />
problem mentioned above. We have designed a standard Pichia system that can be<br />
used for the production of various different vaccines.The system involves an<br />
interchangeable antigen part that is designed based on the variant of the virus<br />
antigen that needs to be produced or any other antigen that needs to be produced. <br />
To illustrate this we present the globular head region of the H1N1 Influenza A virus.</div>Alalanihttp://2010.igem.org/Team:Georgia_State/OursystemTeam:Georgia State/Oursystem2010-10-28T01:14:55Z<p>Alalani: </p>
<hr />
<div>'''Background''':<br />
In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A <br />
virus to the 6 phase alert. This is the highest alert level and it indicates widespread<br />
community transmission of the virus in over two continents. The H1N1 Influenza A virus<br />
is a quadruple reassortment of two swine strains, one human strain, and one avian strain.<br />
More than 214 countries and territories have reported laboratory – confirmed cases of<br />
pandemic H1N1 Influenza A. The US Centers for Disease Control and Prevention reported<br />
between April 2009 and April 2010, approximately 61million cases of pandemic H1N1 occurred,<br />
including 274,000 hospitalizations and 12,470 deaths. <br />
<br />
'''Problem:''' <br />
Efficient and timely vaccine production was a challenge for the scientific<br />
community during the pandemic outbreak of H1N1. Another problem with the <br />
Influenza viruses is the high variability and mutations that they present<br />
with each year. This makes vaccine production time difficult and time consuming.<br />
<br />
'''The Pichia System:'''<br />
The Pichia system that we plan to establish has the capability of overcoming this<br />
problem mentioned above. We have designed a standard Pichia system that can be<br />
used for the production of various different vaccines.The system involves an<br />
interchangeable antigen part that is designed based on the variant of the virus<br />
antigen that needs to be produced or any other antigen that needs to be produced. <br />
To illustrate this we present the globular head region of the H1N1 Influenza A virus.</div>Alalanihttp://2010.igem.org/Team:Georgia_State/OursystemTeam:Georgia State/Oursystem2010-10-28T01:12:40Z<p>Alalani: </p>
<hr />
<div>'''Background''':<br />
In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A virus to the 6 phase alert. This is the highest alert level and it indicates widespread community transmission of the virus in over two continents. The H1N1 Influenza A virus is a quadruple reassortment of two swine strains, one human strain, and one avian strain. More than 214 countries and territories have reported laboratory – confirmed cases of pandemic H1N1 Influenza A. The US Centers for Disease Control and Prevention reported between April 2009 and April 2010, approximately 61million cases of pandemic H1N1 occurred, including 274,000 hospitalizations and 12,470 deaths. <br />
<br />
'''Problem:''' <br />
Efficient and timely vaccine production was a challenge for the scientific community during the pandemic outbreak of H1N1. Another problem with the Influenza viruses is the high variability and mutations that they present with each year. This makes vaccine production time difficult and time consuming.<br />
<br />
'''The Pichia System:'''<br />
The Pichia system that we plan to establish has the capability of overcoming this problem mentioned above. <br />
We have designed a standard Pichia system that can be used for the production of various different vaccines.<br />
The system involves an interchangeable antigen part that is designed based on the variant of the virus antigen that needs to be produced or any other antigen that needs to be produced. <br />
To illustrate this we present the globular head region of the H1N1 Influenza A virus.</div>Alalanihttp://2010.igem.org/Team:Georgia_State/OursystemTeam:Georgia State/Oursystem2010-10-28T01:11:31Z<p>Alalani: New page: '''Background''': In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A virus to the 6 phase alert. This is the highest alert level and it indicates widespre...</p>
<hr />
<div>'''Background''':<br />
In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A virus to the 6 phase alert. This is the highest alert level and it indicates widespread community transmission of the virus in over two continents. The H1N1 Influenza A virus is a quadruple reassortment of two swine strains, one human strain, and one avian strain. More than 214 countries and territories have reported laboratory – confirmed cases of pandemic H1N1 Influenza A. The US Centers for Disease Control and Prevention reported between April 2009 and April 2010, approximately 61million cases of pandemic H1N1 occurred, including 274,000 hospitalizations and 12,470 deaths. <br />
<br />
'''Problem:''' <br />
Efficient and timely vaccine production was a challenge for the scientific community during the pandemic outbreak of H1N1. Another problem with the Influenza viruses is the high variability and mutations that they present with each year. This makes vaccine production time difficult and time consuming.<br />
<br />
'''The Pichia System:'''<br />
The Pichia system that we plan to establish has the capability of overcoming this problem mentioned above. We have designed a standard Pichia system that can be used for the production of various different vaccines. The system involves an interchangeable antigen part that is designed based on the variant of the virus antigen that needs to be produced or any other antigen that needs to be produced. To illustrate this we present the globular head region of the H1N1 Influenza A virus.</div>Alalani