Difference between revisions of "Part:BBa K3866015"

Revision as of 17:03, 25 September 2021

PhaA-RBS-PhaB-RBS-Crt-RBS-Ter GB compatible with B3-B5

The production of butyrate by bacteria can be possible with a set of genes. The superpart 1 includes only four of the nine genes that catalyze the formation of butyric acid. Those are PhaA, PhaB, Crt and Ter genes.

Fig.2:J23115 with RBS

Usage and Biology

First, PhaA and BktB are two β-ketothiolases that carry out the homo-fusion of two acetyl-CoA moieties into acetoacetyl-CoA. Then, the acetoacetyl-CoA is reduced to (S)-3-hydroxybutyryl-CoA (3-HB-CoA) by NADH-dependent Hbd or (R)-3-HB-CoA by NADPH-dependent PhaB. Both (R)-3-HB-CoA and (S)- 3-HB-CoA can be converted to (R)-3-HB and (S)-3-HB, respectively, by a CoA-removing enzyme, such as Pct, TesB, or Ptb/Buk. Alternatively, (R)-3-HB-CoA and (S)-3-HB-CoA can be converted to crotonyl-CoA by PhaJ and Crt, respectively. Crotonyl-CoA is then reduced to butyryl-CoA by NADH-dependent Ter, and subsequently converted to butyrate by one of the above CoA-removing enzymes.

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in pUPD2 BBa_K3505007 and has overhangs compatible for GoldenBraid cloning. The Promoter has position B3-B5.

Fig.1:The overhangs of this part in the GoldenBraid Grammar

Verification and Cloning

Digestion with EcoRV Expected bands at:

4807 bps
1262 bps

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 357
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 357
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 3315
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 357
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 357
Illegal NgoMIV site found at 122
Illegal NgoMIV site found at 226
1000
COMPATIBLE WITH RFC[1000]

Source

Synthesized by Twist Biosciences.

References

i

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3866015 short</partinfo>
-I
+<p>The production of butyrate by bacteria can be possible with a set of genes. The superpart 1 includes only four of the nine genes that catalyze the formation of butyric acid. Those are PhaA, PhaB, Crt and Ter genes.
+[[File:T--Thessaly--AndersonRBS-snap.png|700px|thumb|none|<i><b>Fig.2:</b>J23115 with RBS</i>]]
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
-<!-- -->
+First, PhaA and BktB are two β-ketothiolases  that carry out the homo-fusion of two acetyl-CoA moieties into acetoacetyl-CoA. Then, the acetoacetyl-CoA is reduced to (S)-3-hydroxybutyryl-CoA (3-HB-CoA) by NADH-dependent Hbd or (R)-3-HB-CoA by NADPH-dependent PhaB. Both (R)-3-HB-CoA and (S)- 3-HB-CoA can be converted to (R)-3-HB and (S)-3-HB, respectively, by a CoA-removing enzyme, such as Pct, TesB, or Ptb/Buk.  Alternatively, (R)-3-HB-CoA and (S)-3-HB-CoA can be converted to crotonyl-CoA by PhaJ and Crt, respectively. Crotonyl-CoA is then reduced to butyryl-CoA by NADH-dependent Ter, and subsequently converted to butyrate by one of the above CoA-removing enzymes.
-<span class='h3bb'>Sequence and Features</span>
+===Design Notes===
+The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in pUPD2 BBa_K3505007 and has overhangs compatible for GoldenBraid cloning. The Promoter has position B3-B5.
+[[File:T--Thessaly--GB-GGAG-AATG.jpeg|700px|thumb|none|<i><b>Fig.1:</b>The overhangs of this part in the GoldenBraid Grammar</i>]]
+===Verification and Cloning===
+Digestion with EcoRV
+Expected bands at:
+*4807 bps
+*1262 bps
+===Sequence and Features===
 <partinfo>BBa_K3866015 SequenceAndFeatures</partinfo>
+===Source===
+Synthesized by Twist Biosciences.
-<!-- Uncomment this to enable Functional Parameter display
+===References===
-===Functional Parameters===
+i
-<partinfo>BBa_K3866015 parameters</partinfo>
-<!-- -->