Difference between revisions of "Part:BBa K1590001"

Revision as of 02:39, 19 September 2015

Human Haemoglobin B

Human haemoglobin (hemoglobin) forms a tetramer consisting of two A-chains and two B-chains. This biobrick is a synthetic gene encoding the human haemoglobin B-chain that has been optimized for expression in E. coli.

Usage and Biology

Haemoglobin is the tetrameric protein molecule in red blood cells that carries oxygen. It is composed of four polypeptide chains, which in adults consist of two alpha (A) globin chains and two beta (B) globin chains. The protein also normally contains the iron-containing cofactor haem (or heme). Haemoglobin can still be found free in the blood plasma at a concentration of up to 0.1 g/l.

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iGEM Dundee 2015

This synthetic gene was found to produce stable product when expressed in E. coli cells.

Results

Overexpression and Purification of Haemoglobin B

Initial experiments were carried out to optimize expression of haemoglobin B within our E.coli chassis. We found the following conditions to be optimum: we subcultured 50µl of an overnight culture into fresh LB containing the appropriate antibiotics and grew the cells at 37°C until an OD₆₀₀ of ~0.6 was reached. The production of protein was then induced by adding 1mM (final concentration) of IPTG then the cells were grown for a further 3 hours at 37°C. Next, 1ml of this culture was then taken for analysis by western immunoblot which showed that haemoglobin B was successfully overexpressed. The results from this are illustrated below in Figure 1.

File:Https://static.igem.org/mediawiki/2015/f/fd/Blood-fig2.png

Figure 1: Overexpression of haemoglobin B. Single colonies of M15[pREP4] E. coli containing pQE80-L encoding hHBB were used to inoculate 5ml of fresh LB growth medium supplemented with ampicillin and kanamycin. These overnight cultures were then subcultured and grown at 37°C. Once cultures reached an OD₆₀₀ of 0.6, they were induced with a range of IPTG concentrations. They were left to grow for a further three hours at 37°C, after which 1ml aliquots were pelleted, re-suspended in 100µl of Laemmli buffer and 20µl of these samples were separated by SDS-PAGE (12% acrylamide). This was transferred to a nitrocellulose membrane and probed with an anti-his antibody. Western analysis shows production of of Human Haemoglobin B in E. coli (expected size - 16kDa).

           Purification of haemoglobin beta.

This was then scaled up and 4 litres of E. coli containing pQE80-L hHBB was grown for purification of haemoglobin B by immobilized metal affinity chromatography (IMAC). The optimum conditions for this were as follows: 4 x 1L of fresh LB growth medium containing ampicillin and kanamycin was inoculated with 50ml of haemoglobin beta overnight culture and left to grow until an OD₆₀₀ of between 0.6-1 at 37°C. The expression of protein was then induced by adding 1mM of IPTG then the cells were grown for a further 6 hours at 20°C. Cells were then pelleted and washed in a buffer of 50mM Tris-HCl pH7.5. Cells were then lysed and centrifuged to remove any cell debris. A crude extract was loaded onto a nickel affinity column and the protein was eluted with an imidazole gradient. The results from this can be seen in Figure 2.

thumb|left|500px|Figure 1: Figure 3 - Purification of Human Haemoglobin B by nickel IMAC. A crude extract was loaded onto a nickel affinity column and the protein was eluted with an imidazole gradient (0-1000mM). The fractions corresponding to a peak were collected.

The fractions corresponding to the IMAC peak were retained and concentrated down to 500μl for further purification by size exclusion chromatography (SEC). Figure 3.

Figure 4 - Characterisation of haemoglobin B following SEC (size exclusion chromatography). A) The sample of concentrated fractions containing haemoglobin B from nickel affinity purification was loaded onto an SEC column and the protein was eluted. B) 10µl of each fraction corresponding to the two observed peaks was mixed with 10µl of laemmli buffer and loaded onto an SDS gel. The bands observable on the gel are in line with the expected size of haemoglobin beta - 16kDa. C) Western immunolot was then carried out against an anti-his antibody to confirm the presence of haemoglobin B.

We have succesfully purified haemoglobin B and our next steps were to express and purify its binding partner haptoglobin.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
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COMPATIBLE WITH RFC[12]
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COMPATIBLE WITH RFC[21]
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COMPATIBLE WITH RFC[23]
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COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 22: / Line 22: @@
 <b>Overexpression and Purification of Haemoglobin B</b>
-               <p>Initial experiments were carried out to optimize expression of haemoglobin B within our <i>E.coli</i> chassis. We found the following conditions to be optimum: we subcultured 50µl of an overnight culture into fresh LB containing the appropriate antibiotics and grew the cells at 37°C until an OD<sub>600</sub> of ~0.6 was reached. The production of protein was then induced by adding 1mM (final concentration) of IPTG then the cells were grown for a further 3 hours at 37°C. Next, 1ml of this culture was then taken for analysis by western immunoblot which showed that haemoglobin B was successfully overexpressed. The results from this are illustrated below in Figure 2.</p>
+               <p>Initial experiments were carried out to optimize expression of haemoglobin B within our <i>E.coli</i> chassis. We found the following conditions to be optimum: we subcultured 50µl of an overnight culture into fresh LB containing the appropriate antibiotics and grew the cells at 37°C until an OD<sub>600</sub> of ~0.6 was reached. The production of protein was then induced by adding 1mM (final concentration) of IPTG then the cells were grown for a further 3 hours at 37°C. Next, 1ml of this culture was then taken for analysis by western immunoblot which showed that haemoglobin B was successfully overexpressed. The results from this are illustrated below in Figure 1.</p>
- https://static.igem.org/mediawiki/2015/f/fd/Blood-fig2.png
+[[image: https://static.igem.org/mediawiki/2015/f/fd/Blood-fig2.png|thumb|left|500px|<b>Figure 1: Overexpression of haemoglobin B.</b> Single colonies of M15[pREP4] <i>E. coli</i> containing pQE80-L encoding h<i>HBB</i> were used to inoculate 5ml of fresh LB growth medium supplemented with ampicillin and kanamycin. These overnight cultures were then subcultured and grown at 37°C. Once cultures reached an OD<sub>600</sub> of 0.6, they were induced with a range of IPTG concentrations. They were left to grow for a further three hours at 37°C, after which 1ml aliquots were pelleted, re-suspended in 100µl of Laemmli buffer and 20µl of these samples were separated by SDS-PAGE (12% acrylamide). This was transferred to a nitrocellulose membrane and probed with an anti-his antibody. Western analysis shows production of of Human Haemoglobin B in E. coli (expected size - 16kDa).]]
-                           <p><b>Figure 1 - Overexpression of haemoglobin B.</b> Single colonies of M15[pREP4] <i>E. coli</i> containing pQE80-L encoding h<i>HBB</i> were used to inoculate 5ml of fresh LB growth medium supplemented with ampicillin and kanamycin. These overnight cultures were then subcultured and grown at 37°C. Once cultures reached an OD<sub>600</sub> of 0.6, they were induced with a range of IPTG concentrations. They were left to grow for a further three hours at 37°C, after which 1ml aliquots were pelleted, re-suspended in 100µl of Laemmli buffer and 20µl of these samples were separated by SDS-PAGE (12% acrylamide). This was transferred to a nitrocellulose membrane and probed with an anti-his antibody. Western analysis shows production of of Human Haemoglobin B in E. coli (expected size - 16kDa).</p>
              <b>Purification of haemoglobin beta.</b>
-               <p>This was then scaled up and 4 litres of <i>E. coli</i> containing pQE80-L h<i>HBB</i> was grown for purification of haemoglobin B by immobilized metal affinity chromatography (IMAC).  The optimum conditions for this were as follows: 4 x 1L of fresh LB growth medium containing ampicillin and kanamycin was inoculated with 50ml of haemoglobin beta overnight culture and left to grow until an OD<sub>600</sub> of between 0.6-1 at 37°C. The expression of protein was then induced by adding 1mM of IPTG then the cells were grown for a further 6 hours at 20°C. Cells were then pelleted and washed in a buffer of 50mM Tris-HCl pH7.5. Cells were then lysed and centrifuged to remove any cell debris. A crude extract was loaded onto a nickel affinity column and the protein was eluted with an imidazole gradient. The results from this can be seen in Figure 3.</p>
+               <p>This was then scaled up and 4 litres of <i>E. coli</i> containing pQE80-L h<i>HBB</i> was grown for purification of haemoglobin B by immobilized metal affinity chromatography (IMAC).  The optimum conditions for this were as follows: 4 x 1L of fresh LB growth medium containing ampicillin and kanamycin was inoculated with 50ml of haemoglobin beta overnight culture and left to grow until an OD<sub>600</sub> of between 0.6-1 at 37°C. The expression of protein was then induced by adding 1mM of IPTG then the cells were grown for a further 6 hours at 20°C. Cells were then pelleted and washed in a buffer of 50mM Tris-HCl pH7.5. Cells were then lysed and centrifuged to remove any cell debris. A crude extract was loaded onto a nickel affinity column and the protein was eluted with an imidazole gradient. The results from this can be seen in Figure 2.</p>
+[[ https://static.igem.org/mediawiki/2015/c/ca/Dundeeigem2015purihhbb.png|thumb|left|500px|<b>Figure 1: Figure 3 - Purification of Human Haemoglobin B by nickel IMAC</b>. A crude extract was loaded onto a nickel affinity column and the protein was eluted with an imidazole gradient (0-1000mM). The fractions corresponding to a peak were collected. ]]
- https://static.igem.org/mediawiki/2015/c/ca/Dundeeigem2015purihhbb.png
+ <p>The fractions corresponding to the IMAC peak were retained and concentrated down to 500μl for further purification by size exclusion chromatography (SEC). Figure 3.</p>
-<p><b>Figure 3 - Purification of Human Haemoglobin B by nickel IMAC</b>. A crude extract was loaded onto a nickel affinity column and the protein was eluted with an imidazole gradient (0-1000mM). The fractions corresponding to a peak were collected.  </p>
-              <p>The fractions corresponding to the IMAC peak were retained and concentrated down to 500μl for further purification by size exclusion chromatography (SEC). Figure 4.</p>
                        https://static.igem.org/mediawiki/2015/7/73/Hhbbdundeeigem2015SEC2.jpg