Difference between revisions of "Part:BBa K4604016"

Revision as of 21:26, 10 October 2023

piG_01a (leaky_tetR_bluB)

piG_01a is a construct consisting of the tet promoter/repressor, the modified t7 RBS, a functional bluB gene and the rrnB terminator. The backbone we used in the experiments is pGGAselect. This version on the plasmid is leaky, a modified version of it is uploaded as BioBrick BBa_K4604016.

Usage and Biology

AdoCbl is a bioavailable form of B12. Vitamin B12 is an essential nutrient, humans are dependent on for the production of red blood cells, the synthesis of the DNA and the function of nerves. To form the complete AdoCbl synthesis pathway in E. coli, it would require 28 additional genes. Since this is not realistic nor practical for an iGEM project, we decided on an alternative method. When supplemented with cobinamide (Cbi), a precursor for AdoCbl, E. coli is capable of producing AdoCbl on their own in small amounts. With the overexpression of a naturally occurring gene of the synthesis pathway of sinorhizobium meliloti 2011, called bluB BBa_K4604005, a greater yield can be achieved [2].

Characterization

Western Blot analysis using an anti-His-Tag antibody confirmed the induced expression of BluB. Different concentrations of the inducer doxycycline were tested to identify the optimal yield of the BluB protein.

Figure 1: BluB enzyme production for different inducer concentrations.Detection of recombinantly expressed, his-tagged BluB enzyme with SDS-PAGE followed by Western Blot. Detection of the BluB protein was performed with an anti-his antibody. Loading control: RNA polymerase β-subunit. E. coli BL21 DE3, [piG_01a,BBa_K4604016] overnight culture in LB medium, uninduced.

While we observed an increase in BluB protein yield with increasing inducer concentration, we also noted a leaky expression without induction. This is most likely due to a silent mutation that we introduced in TetR to conform to iGEM standards for this part. This lead to the creation of BBa_K4604016.

Having characterized the expressin of BluB of this BioBrick, next a Liquid Chromatography Mass Spectrometry (LCMS) measurement was performed to confirm its hypothesized ability to produce B12. To estimate the g/g DCW value for the sample with the highest concentration, we measured the cell weight of a 1 mL sample of an independent E. coli BL21(DE3) culture with similar optical density.

Figure 2: OHCbl contents in E. coli BL21(DE3)[piG_01a] cells from production cultures, measured by LC-MS. Colors of bars correspond to respective setups shown in Figure 10. Orange bar respresents measured OHCbl in supernatant of samples. (A) 1mL samples (B) 50 mL samples (C) re-measurement of 50 mL cell pellet and supernatant samples. LC-MS performed at Hannibal Lab, University Medical Center Freiburg. n=1

We successfully confirmed that BluB expression with this BioBrick provides sufficient amounts of DMB for AdoCbl production in E. coli BL21(DE3) from Cbi.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 727
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1647

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-Having characterized the expressin of BluB to our purpose, in this experiment we cultivated <i>E. coli</i> BL21(DE3 containing BBa K4604016 induced and supplemented with Cbi to confirm its hypothesized ability to produce B12 independently of exogenous DMB. Cultures were analyzed for B12 concentrations.
+Having characterized the expressin of BluB of this BioBrick, next a Liquid Chromatography Mass Spectrometry (LCMS) measurement was performed to confirm its hypothesized ability to produce B12. To estimate the g/g DCW value for the sample with the highest concentration, we measured the cell weight of a 1 mL sample of an independent <i>E. coli</i> BL21(DE3) culture with similar optical density.
-<i>E. coli</i> BL21(DE3)[piG_01a] cells from a glycerol stock were grown overnight in 10 mL of LB medium, washed with DPBS, resuspended in 10 mL of M9 medium and then used to inoculate 50 mL cultures in 250 mL shaking flasks to a starting OD600 of 0.1. The cultures were grown until OD600~0.9, then the respective amounts of Cbi and DOX were added to the setups. Initially, cultivation was carried out at 37°C, 200 rpm, before substrate and inducer were added. Afterwards, the temperature for all cultures was reduced to 30°C. OD600 was measured after 0, 26 and 50 hours, LC-MS samples were taken after 26 and 50 hours. The cultivation lasted 50 hours, starting from substrate addition / induction. For detection of B12 concentrations by LC-MS, we took 1 mL of cell culture, centrifuged it, discarded the supernatant and stored the cell pellet at -80°C. Samples were purified and exposed to light deliberately before sending them for external LC-MS analysis, blinded to prevent bias.
-We additionally collected cell pellets of the whole 50 mL cultures as well as the corresponding supernatants after 50 hours for LC-MS analysis.
-To estimate the g/g DCW value for the sample with the highest concentration, we measured the cell weight of a 1 mL sample of an independent <i>E. coli</i> BL21(DE3) culture with similar optical density.
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-By overexpressing BluB, <i>E. coli</i> BL21(DE3)[piG_01a] can produce sufficient amounts of DMB endogenously, leaving DMB supplementation unnecessary regarding B12 production. We successfully confirmed that BluB expression with this BioBrick provides sufficient amounts of DMB for AdoCbl production in <i>E. coli</i> BL21(DE3) from Cbi.
+We successfully confirmed that BluB expression with this BioBrick provides sufficient amounts of DMB for AdoCbl production in <i>E. coli</i> BL21(DE3) from Cbi.