Difference between revisions of "Part:BBa K842003"

Revision as of 12:41, 26 October 2017

fliA

fliA is an alternate sigma factor for the class 3 flagella operons. When transcribed, it forms a component of RNA polymerase sigma 28.

Used for reconstitution of flagella apparatus in E. coli B strains.

32 and 28 belong to the 3rd group of Sigma factors. Sigma factors of that group contain of 2, 3 and 4 areas and structurally are really diverse. 28 is involved in the synthesis of flagell in E.coli(Paget, 2015). For that work of sigma factor the great value has a weaker capacity to the promoter melting. It has been shown that having the strict consensus in the promoter region is important for 28 , it provides a strict specifity of recognition in the initiation of transcription(Koo et al, 2009).

Shown consensus:

(doi:10.1093/nar/gkm456)

It may be explained with that fact, that this type of sigma factors may not have one or several amino acid residues, required for villiany -11A when you initiate promoter region melting(Feklístov et al, 2014).

Pic. 1. Alignment of amino acid sequences of the alternative Sigma relative to the main one. Area 3. 2 is shown in red.

(doi:10.1093/nar/gkt1384)

Cloning and Uses: This part was generated via PCR from the E. coli strain DH5α and cloned into the vector pSB1C3. It is to be used in conjunction with the entire flagella synthesis genetic pathway to reproduce a complete flagella apparatus.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 3
Illegal PstI site found at 91
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 3
Illegal PstI site found at 91
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 3
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 3
Illegal PstI site found at 91
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 3
Illegal PstI site found at 91
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 426
Illegal SapI.rc site found at 451
Illegal SapI.rc site found at 607

@@ Line 5: / Line 5: @@
 Used for reconstitution of flagella apparatus in E. coli B strains.
+32 and 28 belong to the 3rd group of Sigma factors. Sigma factors of that group contain of 2, 3 and 4 areas and structurally are really diverse. 28 is involved in the synthesis of flagell in E.coli(Paget, 2015). For that work of sigma factor the great value has a weaker capacity to the promoter melting. It has been shown that having the strict consensus in the promoter region is important for 28 , it provides a strict specifity of recognition in the initiation of transcription(Koo et al, 2009).
+Shown consensus:
+[[File:consensus.jpg|options|caption]]
+[https://www.ncbi.nlm.nih.gov/pubmed/17576668 (doi:10.1093/nar/gkm456)]
+It may be explained with that fact, that this type of sigma factors may not have one or several amino acid residues, required for villiany -11A when you initiate promoter region melting(Feklístov et al, 2014).
+[[File:Alignment.jpg|options|caption]]
+Pic. 1. Alignment of amino acid sequences of the alternative Sigma relative to the main one. Area 3. 2 is shown in red.
+[https://www.ncbi.nlm.nih.gov/pubmed/?term=10.1093%2Fnar%2Fgkt1384 (doi:10.1093/nar/gkt1384)]
 Cloning and Uses:
@@ Line 18: / Line 33: @@
 <!-- Add more about the biology of this part here
 ===Usage and Biology===
+During transcription setting with the participation of the Sigma - subunit, it’s necessary to pay attention to the factors that can product yield(in this case, RNA transcript). The main factors affecting the enzyme are the salt concentration in the buffer reaction and the temperature of the experiment.
+Standard conditions for carrying out the transcription is considered the holding in buffer containing 40-50 mM of NaCl. However, other concentrations and other salts were checked and it was found out that if you use KCl instead of NaCl reaction optium doesn’t shifts and remains at concentration of 40-50 mM. But during using glutamate and sodium acetate instead of chloride, the reaction optium shifts to the region of high concentrations(of 300-400 mM). The contribution of temperature to the efficiency of the reaction is also worth considering.
+Moreover, it should be noted that operation optium of RNA polymerase containing s70 - subunit is around 37 degrees Celsius, while the enzyme conteining s28 - subunit, this value decreases to 30 degrees Celsius(for achieving  maximum efficiency of transcription reaction). (Promoter Selectivity of Escherichia coli RNA Polymerase sF Holoenzyme Involved in Transcription of Flagellar and Chemotaxis Genes TAPAS KUMAR KUNDU,1 SHUICHI KUSANO,1,2 AND AKIRA ISHIHAMA1 *).
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