Difference between revisions of "Part:BBa K3771075"

Revision as of 14:17, 18 October 2021

Ptrc-CDO1

Description

P_trc-cdo1 is a composite part consisting of the trc promoter and the cdo1 sequences. This part was used in in vivo testing of taurine production. The sequence for cdo1 and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).

Biology

Trc promoter constitutively facilitates the expression of CDO1 enzyme.

Fig. 1 Confirmation of cdo1 fragment by PCR. M: Marker; Lane 1: cdo1 (603 bp)

Fig. 2 Confirmation of pSB4KI-Ptrc-CDO1 by digestion. M: Marker; Lane 1~3: Different colonies of pSB4KI-Ptrc-CDO1 (5470 bp)

Fig. 3 Transformation / CDO1 in DH5α

Fig. 4 Confirmation of protein expression of CDO1. M: Marker; Lane1: CDO1 in DH5α without induction; Lane2: CDO1 in DH5α with induction (~22 kDa)

Usage

CDO1 was used in in vivo testing of taurine production. The sequence for CDO1 enzyme and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).

Fig. 5 Taurine production of Ptrc-cdo1 +PlacI-csad +Ptrc-cs in different growth mediums.

Fig. 6 Taurine production of Ptrc-cdo1+Ptrc-csad in different growth mediums.

Fig. 7 Taurine production (extracellular) of CDO1 and CSAD in ∆tauD.

Fig. 8 Taurine production (extracellular) of CDO1 and CSAD in DkO.

Fig. 9 Taurine production (intracellular and extracellular) of CDO1 and CSAD in DkO and top10.

Fig. 10 Taurine production (extracellular) of CDO1 and CSAD with expression of CSAD regulated by soxS promoter in DkO.

Characterization

In Vivo Test 1: Choosing a Growth Medium Since the intestine is not as nutrient-rich as LB (lysogeny broth), we chose to test taurine production in both LB and M9 (minimal salts) mediums. M9 consists of fewer nutrients than LB and more closely mimics the nutrient composition of the human intestine. First, Ptrc-cdo1 (BBa_K3771075), PlacI-csad (BBa_K3771076), and Ptrc-cs (BBa_K3771078) were transformed into DH5α strain. In another sample, only Ptrc-cdo1 and Ptrc-csad (BBa_K3771077) were transformed. The two samples were each grown in LB with IPTG inducer (LB+), M9 with IPTG inducer (M9+) , and M9 without IPTG inducer (M9-) mediums. The samples’ respective growth curves are shown in figure 21 and 22. Interestingly, the sample with all three enzymes (Ptrc-cdo1 +PlacI-csad +Ptrc-cs) had a lower growth rate in M9+ medium than in M9- medium. In the sample with two enzymes (Ptrc-cdo1+Ptrc-csad), M9 medium with or without an inducer added produced similar growth curves. Both samples grew best in LB (Fig. 11, 12)

Growth curve of Ptrc-cdo1 +PlacI-csad +Ptrc-cs in different growth mediums.

Growth curve of Ptrc-cdo1+Ptrc-csad in different growth mediums.

References

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 <br>In Vivo Test 1: Choosing a Growth Medium
 Since the intestine is not as nutrient-rich as LB (lysogeny broth), we chose to test taurine production in both LB and M9 (minimal salts) mediums. M9 consists of fewer nutrients than LB and more closely mimics the nutrient composition of the human intestine.
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   <div style="width=100%; display:flex; align-items: center; justify-content: center;">
-  <img src="https://2021.igem.org/wiki/images/6/6b/T--NCKU_Tainan--invivo1.png" style="width:35%;">
+  <img src="https://2021.igem.org/wiki/images/c/c9/T--NCKU_Tainan--invivo-growth.png" style="width:35%;">
   </div>
   <p>Growth curve of Ptrc-cdo1+Ptrc-csad in different growth mediums.</p>
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 ===Usage and Biology===
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+<!-- --
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K3771075 SequenceAndFeatures</partinfo>