Difference between revisions of "Part:BBa K1590001"

Revision as of 21:05, 18 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 Beta

Initial experiments were carried out to optimise expression of haemoglobin beta (hHBB) within our E.coli chassis. The synthetic gene was subcloned into the pQE80-L overexpression vector that adds an N-terminal hexa-histidine tag onto the N-terminus of the protein. We found the following to be the optimum conditions:

We subcultured 50 µl of an overnight culture into fresh LB containing the appropriate antibiotics and grew the cells at 37C until an OD-600 of ~0.6 was reached. The production of protein was then induced by adding 1 mM (final concentration)of IPTG then the cells were grown for a further 3 hours at 37C. Next, 1 ml of this culture was then taken for analysis by western immunblot which showed that haemoglobin beta was successfully overexpressed (Figure 1).

File:Dundee15 hHGB.jpg

Figure 1: Western anakysis of Human Haemoglobin B production in E. coli.

Sequence and Features

Assembly Compatibility:

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

@@ Line 20: / Line 20: @@
 ===Results===
+<b>Overexpression and Purification of Haemoglobin Beta</b>
+Initial experiments were carried out to optimise expression of haemoglobin beta (hHBB) within our <i>E.coli</i> chassis. The synthetic gene was subcloned into the pQE80-L overexpression vector that adds an N-terminal hexa-histidine tag onto the N-terminus of the protein. We found the following to be the optimum conditions:
+We subcultured 50 µl of an overnight culture into fresh LB containing the appropriate antibiotics and grew the cells at 37C until an OD-600 of ~0.6 was reached. The production of protein was then induced by adding 1 mM (final concentration)of IPTG then the cells were grown for a further 3 hours at 37C. Next, 1 ml of this culture was then taken for analysis by western immunblot which showed that haemoglobin beta was successfully overexpressed (Figure 1).
+[[Image:Dundee15_hHGB.jpg]]
+:Figure 1: Western anakysis of Human Haemoglobin B production in <i>E. coli</i>.