Difference between revisions of "Part:BBa K2762006"

Latest revision as of 14:22, 17 October 2018

PT7-B0034-rbcX-B0034-rbcS-B0015

Background

The rbcXS part encode the RbcS subunit of RubisCo and the chaperon RbcX. The RbcS affect the activity of RubisCO and RbcX contribute to the correct folding of the whole RubisCO enzyme.

Mechanism

The rbcX and rbcS genes are from cyanobacteria Synechococcus elongatus PCC7002. We designed the T7 promoter (BBa_I719005) for the gene and cloned gene in BL21 to ensure the high expression rate. It has reported that the expression of RubisCO could not be detectable while only add single promoter of the RbcL-RbcX-RbcS gene fragment. Therefore we added another T7 promoter on the upstream of rbcX-rbcS gene and named the part as rbcXS. We also did the coden optimization for the gene to ensure successful expression. The activation of RubisCO will be maximize after the binding of the RbcS subunit. To get more information, see our composite part: BBa_K2762011

Characterization

Expression in E. coli

We did two experiment to confirm the expression of this part. First we inserted the part on pSB1C3 and cloned the plasmid into DH5 alpha. We extracted the plasmid after the colony formed and did the enzyme digestion to confirm the insertion was successful. Second, we cloned the plasmid into BL21(DE3) and induced the promoter with IPTG. We then did the SDS-PAGE to confirm the present of the RbcX and RbcS protein.

T--NCKU Tainan--part BBa K2762006 new.png

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]

References

[1] Janet Newman 1t and Steven Gutteridge2*. (1994,JUN.15). Structure of an effector-induced inactivated state of ribulose 1,5-bisphosphate carboxylase/oxygenase: the binary complex between enzyme and xylulose 1,5-bisphosphate. Cell

[2] Inger Andersson ^a,*, Anders Backlund ^b. (2007, DEC.20). Structure and function of Rubisco. Plant Physiology and Biochemistry.

[3] Fuyu Gong, Guoxia Liu, Xiaoyun Zhai,Jie Zhou, Zhen Cai and Yin Li1 .(2015,Jun 18). Quantitative analysis of an engineered CO₂-fixing Escherichia coli reveals great potential of heterotrophic CO₂ fixation. Biotechnology for Biofuels.

[4] Sandra Saschenbrecker,^1,2 Andreas Bracher,^1,2,* Karnam Vasudeva Rao,¹ Bharathi Vasudeva Rao,¹ F. Ulrich Hartl,^1,* and Manajit Hayer-Hartl^1,*. (2007, APR. 25). Structure and Function of RbcX, an Assembly Chaperone for Hexadecameric Rubisco. Cell

[5] Cuimin Liu^1*, Anna L. Young^2*, Amanda Starling-Windhof¹, Andreas Bracher¹, Sandra Saschenbrecker¹, Bharathi Vasudeva Rao¹, Karnam Vasudeva Rao¹, Otto Berninghausen², Thorsten Mielke³, F. Ulrich Hartl¹, Roland Beckmann² & Manajit Hayer-Hartl¹. (2010, JAN. 4). Coupled chaperone action in folding and assembly of hexadecameric Rubisco. Nature

@@ Line 2: / Line 2: @@
 __NOTOC__
 <partinfo>BBa_K2762006 short</partinfo>
+===Background===
+The <i>rbcXS</i> part encode the RbcS subunit of RubisCo and the chaperon RbcX. The RbcS affect the activity of RubisCO and RbcX contribute to the correct folding of the whole RubisCO enzyme.
--
+===Mechanism===
+The <i>rbcX</i> and <i>rbcS</i> genes are from cyanobacteria <i>Synechococcus elongatus</i> PCC7002. We designed the T7 promoter (BBa_I719005) for the gene and cloned gene in BL21  to ensure the high expression rate. It has reported that the expression of RubisCO could not be detectable while only add single promoter of the RbcL-RbcX-RbcS gene fragment. Therefore we added another T7 promoter on the upstream of <i>rbcX-rbcS</i> gene and named the part as <i>rbcXS</i>. We also did the coden optimization for the gene to ensure successful expression. The activation of RubisCO will be maximize after the binding of the RbcS subunit. To get more information, see our composite part: BBa_K2762011
+==Characterization==
+===Expression in <i>E. coli</i>===
+We did two experiment to confirm the expression of this part. First we inserted the part on pSB1C3 and cloned the plasmid into DH5 alpha. We extracted the plasmid after the colony formed and did the enzyme digestion to confirm the insertion was successful. Second, we cloned the plasmid into BL21(DE3) and induced the promoter with IPTG. We then did the SDS-PAGE to confirm the present of the RbcX and RbcS protein.
+[[File:T--NCKU Tainan--part BBa K2762006 new.png|200px|centre]]
 <!-- Add more about the biology of this part here
@@ Line 17: / Line 25: @@
 <partinfo>BBa_K2762006 parameters</partinfo>
 <!-- -->
+====References====
+[1] Janet Newman 1t and Steven Gutteridge2*. (1994,JUN.15). Structure of an effector-induced inactivated state of ribulose 1,5-bisphosphate carboxylase/oxygenase: the binary complex between enzyme and xylulose 1,5-bisphosphate.<i> Cell</i>
+[2] Inger Andersson <sup>a,</sup>*, Anders Backlund <sup>b</sup>. (2007, DEC.20). Structure and function of Rubisco. <i>Plant Physiology and Biochemistry</i>.
+[3] Fuyu Gong, Guoxia Liu, Xiaoyun Zhai,Jie Zhou, Zhen Cai and Yin Li1 .(2015,Jun 18). Quantitative analysis of an engineered CO<sub>2</sub>-fixing Escherichia coli reveals great potential of heterotrophic CO<sub>2</sub> fixation.<i> Biotechnology for Biofuels.</i>
+[4] Sandra Saschenbrecker,<sup>1,2</sup> Andreas Bracher,<sup>1,2,*</sup> Karnam Vasudeva Rao,<sup>1</sup> Bharathi Vasudeva Rao,<sup>1</sup> F. Ulrich Hartl,<sup>1,*</sup> and Manajit Hayer-Hartl<sup>1,*</sup>. (2007, APR. 25). Structure and Function of RbcX, an Assembly Chaperone for Hexadecameric Rubisco. <i>Cell</i>
+[5] Cuimin Liu<sup>1*</sup>, Anna L. Young<sup>2*</sup>, Amanda Starling-Windhof<sup>1</sup>, Andreas Bracher<sup>1</sup>, Sandra Saschenbrecker<sup>1</sup>, Bharathi Vasudeva Rao<sup>1</sup>, Karnam Vasudeva Rao<sup>1</sup>, Otto Berninghausen<sup>2</sup>, Thorsten Mielke<sup>3</sup>, F. Ulrich Hartl<sup>1</sup>, Roland Beckmann<sup>2</sup> & Manajit Hayer-Hartl<sup>1</sup>. (2010, JAN. 4). Coupled chaperone action in folding and assembly of hexadecameric Rubisco. <i>Nature</i>