IGEM:Brown/2007/Sensor/What to detect?

Bacterial signal transduction network in a genomic perspective

author: Michael Y. Galperin

Tables 1 and 2 show what types of signalling molecules are present in different types of prokaryotes.

It looks like S_TKc (Serine-Threonine kinase, catalytic) would work best for E. Coli because there are not many of them in E. Coli naturally. Or, we may want to use a signalling protein that doesn't exist yet in E. Coli, to prevent confusion and false signals.

Signal transduction: Hair brains in bacterial chemotaxis

authors: Jeff Stock and Mikhail Levit

In their ‘ON’ state, in the absence of attractants, several receptors bind to CheA in such a way that CheA is activated over 100-fold [24], and conversely CheA binding to the receptors appears to be required for long-range structural interactions that serve to organize the array. In the ‘OFF’ state, in the presence of attractants, it is as if the CheA dimer is torn apart by binding to the receptor network.

In E. coli, at least five different receptors — Tar, Tsr, Tap, Trg and Aer — appear to be intermingled within the same complex.

a) Tar: aspartate and maltose; cobalt and nickel

b) Tsr: serine

c) Tap: Taxis towards peptides

d) Trg: ribose and galactose

e) Aer: directs taxis towards ribose, galactose, maltose, malate, proline and alanine

a. CheA is a kinase that takes a phosphate off of ATP and attaches it to itself

b. CheW connects CheA to the chemoreceptor

Binding Proteins

author: Dr. Leonidas G. Bachas

Sensing System for Zinc Based on Zinc-Binding Protein

Zinc is an essential element in our diet. Too little zinc can cause problems, but too much zinc is also harmful. Severe soil zinc deficiency can cause complete crop failure. Certain microorganisms are known to survive in highly toxic environments contaminated with toxic species such as mercury, arsenic, cadmium, zinc, lead, copper or nickel. Resistance is associated with presence of resistance operons which are precisely regulated. Operon consists of gene for regulatory protein to which toxic metals binds and induce the expression of other genes of operon. Detoxification occurs either by pumping the toxic metals out of the cell or by expression of metallothionein, a cysteine rich protein that chelates heavy metals. We plan to take advantage of this specific binding between the regulatory protein and the toxic species in order to develop a sensing system for the target toxic analyte. In this project we have replaced the genes of the operon for zinc resistance with the genes that encode for reporter proteins to develop a biosensor for zinc.

Detection of Sulfate Using Periplasmic Sulfate-Binding Protein

Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe, which allow the development of reagentless sensing systems for their corresponding ligands. On the basis of this strategy we have evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe for rational designe of a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. Different environment-sensitive fluorescent probes were then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed.

An Exceptionally Selective Lead(ii)-Regulatory Protein from Ralstonia Metallidurans: Development of a Fluorescent Lead(ii) Probe

Lead Detection Paper (THIS IS AWESOME!)

Supplement to the Lead Detection Paper (mentions work done in ecoli)

Can ecoli survive in lead?

PbrR protein [Ralstonia metallidurans CH34]

Other Aliases: pMOL30_092

Genomic context: Plasmid pMOL30 (Plasmid 1)

Annotation: NC_006466.1 (114932..115370)

GeneID: 3170418

MNIQIGELAKRTACPVVTIRFYEQEGLLPPPGRSRGNFRLYGEEHVERLQFIRHCRSLDMPLSDVRTLLS YRKRPDQDCGEVNMLLDEHIRQVESRIGALLELKHHLVELREACSGARPAQSCGILQGLSDCVCDTRGTT AHPSD

114933..115370 (including stop codon)

DNA sequence:

atgaatat ccagatcggc gagcttgcca agcgcaccgc atgcccggtg gtgaccattc gcttctacga acaagaaggg ctgttgccgc cgccgggccg cagccggggg aattttcgcc gtatggcga ggagcacgtg gagcgcttgc agttcattcg tcactgccgg tctctggata tgccgttgag cgacgtacgg accttattga gttaccggaa gcggcccgac caggattgcg tgaagtcaa tatgctcttg gatgagcaca tccgtcaggt cgaatctcgg atcggagctt tgctcgaact gaagcaccat ttggtggaac tgcgcgaagc ctgttctggt gccaggcccg ccaatcgtg cgggattctg cagggactgt cggactgcgt gtgtgatacg cgggggacca ccgcccatcc aagcgactag

PstI site found in the sequence at base pair 373.

Lead Promoter Sequence:

GGTTGCTTCCTATAAAAAACTTGACTCTATATCTACTAGAGGTTTTCTAATGATGGCATC CGGGGAAAACCTTGTCAATGAAGAGCGATCTATG

Primers Ordered (that actually work and will be kept):

PbrR691 5' Primer a: GTTTCTTCGAATTCGCGGCCGCTTCTAGatgaatatccagatcggcgag

PbrR691 5' Primer b: GTTTCTTCGAATTCGCGGCCGCTTCTAGatgaatatccagatcgg

PbrR691 5' Primer c: GTTTCTTCGAATTCGCGGCCGCTTCTAGatgaatatccagatcggcgagcttg

Primers Ordered July 2, 2007:

PbrR691 3' Primer: GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTACTAGTCGCTTGGATGGGC

Promoter 5' Primer: GTTTCTTCGAATTCGCGGCCGCTTCTAGAGccgcatcatggttgcttcc

Promoter 3' Primer: GTTTCTTCCTGCAGCGGCCGCTACTAGTACATAGATGATCGCTCTTCATTGACAAGG

PbrR691 Sequence Analysis to remove Pst1 restriction site: Pst1 Restriction Site:

5'CTGCAG

3'GACGTC

5'---CTGCA G---3'

3'---G ACGTC---5'

1. atg Met start

2. aat Asn

3. atc Ile

4. cag

5. atc

6. ggc

7. gag

8. ctt

9. gcc

10. aag

11. cgc

12. acc

13. gca

14. tgc

15. ccg

16. gtg

17. gtg

18. acc

19. att

20. cgc

21. ttc

22. tac

23. gaa

24. caa

25. gaa

26. ggg

27. ctg

28. ttg

29. ccg

30. ccg

31. ccg

32. ggc

33. cgc

34. agc

35. cgg

36. ggg

37. aat

38. ttt

39. cgc

40. cgt

41. atg

42. gcg

43. agg

44. agc

45. acg

46. tgg

47. agc

48. gct

49. tgc

50. agt

51. tca

52. ttc

53. gtc

54. act

55. gcc

56. ggt

57. ctc

58. tgg

59. ata

60. tgc

61. cgt

62. tga

63. gcg

64. acg

65. tac

66. gga

67. cct

68. tat

69. tga

70. gtt

71. acc

72. gga

73. agc

74. ggc

75. ccg

76. acc

77. agg

78. att

79. gcg

80. tga

81. agt

82. caa

83. tat

84. gct

85. ctt

86. gga

87. tga

88. gca

89. cat

90. ccg

91. tca

92. ggt

93. cga

94. atc

95. tcg

96. gat

97. cgg

98. agc

99. ttt

100. gct

101. cga

102. act

103. gaa

104. gca

105. cca

106. ttt

107. ggt

108. gga

109. act

110. gcg

111. cga

112. agc

113. ctg

114. ttc

115. tgg

116. tgc

117. cag

118. gcc

119. cgc

120. caa

121. tcg

122. tgc

123. ggg

124. att

125. ctg Leu: ctt (9.7), ctc (10.4), cta (3.09), ctg (52.8)

126. cag Gln: caa (33.4), cag (66.6)

127. gga

128. ctg

129. tcg

130. gac

131. tgc

132. gtg

133. tgt

134. gat

135. acg

136. cgg

137. ggg

138. acc

139. acc

140. gcc

141. cat

142. cca

143. agc

144. gac

145. tag

PstI site found in the sequence at base pair 373.

1. Methionine (atg → aug)

2. Asparagine (aat → aau)

3. Isoleucine (atc → auc)

4. Glutamine (cag → cag)

5. Isoleucine (atc → auc)

6. Glycine (ggc → ggc)

7. Glutamic acid (gag → gag)

8. Leucine (ctt → cuu)

9. Alanine (gcc → gcc)

10. Lysine (aag → aag)

11. Arginine (cgc → cgc)

12. Threonine (acc → acc)

13. Alanine (gca → gca)

14. Cysteine (tgc → ugc)

15. Proline (ccg → ccg)

16. Valine (gtg → gug)

17. Valine (gtg → gug)

18. Threonine (acc → acc)

19. Isoleucine (att → auu)

20. Arginine (cgc → cgc)

21. Phenylalanine (ttc → uuc)

22. Tyrosine (tac → uac)

23. Glutamic acid (gaa → gaa)

24. Glutamine (caa → caa)

25. Glutamic acid (gaa → gaa)

26. Glycine (ggg → ggg)

27. Leucine (ctg → cug)

28. Leucine (ttg → uug)

29. Proline (ccg → ccg)

30. Proline (ccg → ccg)

31. Proline (ccg → ccg)

32. Glycine (ggc → ggc)

33. Arginine (cgc → cgc)

34. Serine (agc → agc)

35. Arginine (cgg → cgg)

36. Glycine (ggg → ggg)

37. Asparagine (aat → aau)

38. Phenylalanine (ttt → uuu)

39. Arginine (cgc → cgc)

40. Arginine (cgt → cgu)

41. Methionine (atg → aug)

42. Alanine (gcg → gcg)

43. Arginine (agg → agg)

44. Serine (agc → agc)

45. Threonine (acg → acg)

46. Tryptophan (tgg → ugg)

47. Serine (agc → agc)

48. Alanine (gct → gcu)

49. Cysteine (tgc → ugc)

50. Serine (agt → agu)

51. Serine (tca → uca)

52. Phenylalanine (ttc → uuc)

53. Valine (gtc → guc)

54. Threonine (act → acu)

55. Alanine (gcc → gcc)

56. Glycine (ggt → ggu)

57. Leucine (ctc → cuc)

58. Tryptophan (tgg → ugg)

59. Isoleucine (ata → aua)

60. Cysteine (tgc → ugc)

61. Arginine (cgt → cgu)

62. stop (tga → uga)

63. Alanine (gcg → gcg)

64. Threonine (acg → acg)

65. Tyrosine (tac → uac)

66. Glycine (gga → gga)

67. Proline (cct → ccu)

68. Tyrosine (tat → uau)

69. stop (tga → uga)

70. Valine (gtt → guu)

71. Threonine (acc → acc)

72. Glycine (gga → gga)

73. Serine (agc → agc)

74. Glycine (ggc → ggc)

75. Proline (ccg → ccg)

76. Threonine (acc → acc)

77. Arginine (agg → agg)

78. Isoleucine (att → auu)

79. Alanine (gcg → gcg)

80. stop (tga → uga)

81. Serine (agt → agu)

82. Glutamine (caa → caa)

83. Tyrosine (tat → uau)

84. Alanine (gct → gcu)

85. Leucine (ctt → cuu)

86. Glycine (gga → gga)

87. stop (tga → uga)

88. Alanine (gca → gca)

89. Histidine (cat → cau)

90. Proline (ccg → ccg)

91. Serine (tca → uca)

92. Glycine (ggt → ggu)

93. Arginine (cga → cga)

94. Isoleucine (atc → auc)

95. Serine (tcg → ucg)

96. Aspartic acid (gat → gau)

97. Arginine (cgg → cgg)

98. Serine (agc → agc)

99. Phenylalanine (ttt → uuu)

100. Alanine (gct → gcu)

101. Arginine (cga → cga)

102. Threonine (act → acu)

103. Glutamic acid (gaa → gaa)

104. Alanine (gca → gca)

105. Proline (cca → cca)

106. Phenylalanine (ttt → uuu)

107. Glycine (ggt → ggu)

108. Glycine (gga → gga)

109. Threonine (act → acu)

110. Alanine (gcg → gcg)

111. Arginine (cga → cga)

112. Serine (agc → agc)

113. Leucine (ctg → cug)

114. Phenylalanine (ttc → uuc)

115. Tryptophan (tgg → ugg)

116. Cysteine (tgc → ugc)

117. Glutamine (cag → cag)

118. Alanine (gcc → gcc)

119. Arginine (cgc → cgc)

120. Glutamine (caa → caa)

121. Serine (tcg → ucg)

122. Cysteine (tgc → ugc)

123. Glycine (ggg → ggg)

124. Isoleucine (att → auu)

125. Leucine (ctg → cug)

126. Glutamine (cag → cag)

127. Glycine (gga → gga)

128. Leucine (ctg → cug)

129. Serine (tcg → ucg)

130. Aspartic acid (gac → gac)

131. Cysteine (tgc → ugc)

132. Valine (gtg → gug)

133. Cysteine (tgt → ugu)

134. Aspartic acid (gat → gau)

135. Threonine (acg → acg)

136. Arginine (cgg → cgg)

137. Glycine (ggg → ggg)

138. Threonine (acc → acc)

139. Threonine (acc → acc)

140. Alanine (gcc → gcc)

141. Histidine (cat → cau)

142. Proline (cca → cca)

143. Serine (agc → agc)

144. Aspartic acid (gac → gac)

145. stop (tag → uag)

July 3, 2007: New Developments - Email from Dr. van der Niels

PbrR691 Sequence Analysis according to GenBank: atgaatat ccagatcggc gagcttgcca agcgcaccgc atgcccggtg gtgaccattc gcttctacga acaagaaggg ctgttgccgc cgccgggccg cagccggggg aattttcgcc gtatggcga ggagcacgtg gagcgcttgc agttcattcg tcactgccgg tctctggata tgccgttgag cgacgtacgg accttattga gttaccggaa gcggcccgac caggattgcg tgaagtcaa tatgctcttg gatgagcaca tccgtcaggt cgaatctcgg atcggagctt tgctcgaact gaagcaccat ttggtggaac tgcgcgaagc ctgttctggt gccaggcccg ccaatcgtg cgggattctg cagggactgt cggactgcgt gtgtgatacg cgggggacca ccgcccatcc aagcgactag

1 atgaatatcc agatcggcga gcttgccaag cgcaccgcat gcccggtggt gaccattcgc 61 ttctacgaac aagaagggct gttgccgccg ccgggccgca gccgggggaa ttttcgcctg 121 tatggcgagg agcacgtgga gcgcttgcag ttcattcgtc actgccggtc tctggatatg 181 ccgttgagcg acgtacggac cttattgagt taccggaagc ggcccgacca ggattgcggt 241 gaagtcaata tgctcttgga tgagcacatc cgtcaggtcg aatctcggat cggagctttg 301 ctcgaactga agcaccattt ggtggaactg cgcgaagcct gttctggtgc caggcccgcc 361 caatcgtgcg ggattctgca gggactgtcg gactgcgtgt gtgatacgcg ggggaccacc 421 gcccatccaa gcgactag

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NC_006466.1&from=114933&to=115370&dopt=gb

PbrR691 Sequence According to Fasta:

ATGAATATCCAGATCGGCGAGCTTGCCAAGCGCACCGCATGCCCGGTGGTGACCATTCGCTTCTACGAAC AAGAAGGGCTGTTGCCGCCGCCGGGCCGCAGCCGGGGGAATTTTCGCCTGTATGGCGAGGAGCACGTGGA GCGCTTGCAGTTCATTCGTCACTGCCGGTCTCTGGATATGCCGTTGAGCGACGTACGGACCTTATTGAGT TACCGGAAGCGGCCCGACCAGGATTGCGGTGAAGTCAATATGCTCTTGGATGAGCACATCCGTCAGGTCG AATCTCGGATCGGAGCTTTGCTCGAACTGAAGCACCATTTGGTGGAACTGCGCGAAGCCTGTTCTGGTGC CAGGCCCGCCCAATCGTGCGGGATTCTGCAGGGACTGTCGGACTGCGTGTGTGATACGCGGGGGACCACC GCCCATCCAAGCGACTAG

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NC_006466.1&from=114933&to=115370&dopt=fasta

Inverse complement of above sequence:

CTAGTCGCTTGGATGGGCGGTGGTCCCCCGCGTATCACACACGCAGTCCGACAGTCCCTG CAGAATCCCGCACGATTGGGCGGGCCTGGCACCAGAACAGGCTTCGCGCAGTTCCACCAA ATGGTGCTTCAGTTCGAGCAAAGCTCCGATCCGAGATTCGACCTGACGGATGTGCTCATC CAAGAGCATATTGACTTCACCGCAATCCTGGTCGGGCCGCTTCCGGTAACTCAATAAGGT CCGTACGTCGCTCAACGGCATATCCAGAGACCGGCAGTGACGAATGAACTGCAAGCGCTC CACGTGCTCCTCGCCATACAGGCGAAAATTCCCCCGGCTGCGGCCCGGCGGCGGCAACAG CCCTTCTTGTTCGTAGAAGCGAATGGTCACCACCGGGCATGCGGTGCGCTTGGCAAGCTC GCCGATCTGGATATTCAT

438 bases  146 aa

PbrR691 Protein Sequence According to NCBI: MNIQIGELAKRTACPVVTIRFYEQEGLLPPPGRSRGNFRLYGEEHVERLQFIRHCRSLDMPLSDVRTLLSYRKRPDQDCGEVNMLLDEHIRQVESRIGALLELKHHLVELREACSGARPAQSCGILQGLSDCVCDTRGTTAHPSD

145

According to Niels: “Please find below the correct sequence of the PbrR691 gene. This sequence is the inverse complement and begins at the STOP codon from pbrR691. Also indicated is the ATG start codon for pbrR691 (bold, underlined). The ATG at the end is the start of the pbrA691 gene.

TCAGGCGGGCTCGGCCAAGCTGTTGAGAATGCCGCACTCGCGCGAGGTTCGGGCGCTATCGCAGGAGCGTCGCAGATCCATTAACTCGCGCTCCAAGGCGCGCAATTCCTTCATCTTGGTCCGCACTTGCGCGATATGAGCGTCGACCAGCGCGTTCACCTCGCCGCAGCCCAACTCTGGCCGATCCCGTAAGTTCAGCAGTTGACGGATCTCATCCAGCGTCATGTCCTTCGCCCGGCAGCGGCGGATGAACAGCAAGCGCTGCAAATGGACTTCGTCATAGAGCCTGAAGTTGCCCTCGCTACGTGCAGGCTCGGGCAGCAAGCCTTCTGACTCGTAAAAGCGCACGGTCTGCACCAAGCAATCTGCCTTCTTGCCCAGTTCACCGATCCGCATCATGGTTGCTTCCTATAAAAAACTTGACTCTATATCTACTAGAGGTTTTCTAATGATGGCATCCGGGGAAAACCTTGTCAATGAAGAGCGATCTATG

493  164.3 aa

399  133 aa

There are no PstI sites in this sequence.

In order to make a gene fusion that is under control of PbrR691 you will need to place your reporter gene downstream of the ATG start codon at the end of the is sequence.”

What this means: Gene orientation according to PubMed:

plasmid: pMOL30

http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=Retrieve&dopt=full_report&list_uids=3170418#refseq

Inverse of above sequence:

GTATCTAGCGAGAAGTAACTGTTCCAAAAGGGGCCTACGGTAGTAATCTTTTGGAGATCA TCTATATCTCAGTTCAAAAAATATCCTTCGTTGGTACTACGCCTAGCCACTTGACCCGTT CTTCCGTCTAACGAACCACGTCTGGCACGCGAAAATGCTCAGTCTTCCGAACGACGGGCT CGGACGTGCATCGCTCCCGTTGAAGTCCGAGATACTGCTTCAGGTAAACGTCGCGAACGA CAAGTAGGCGGCGACGGCCCGCTTCCTGTACTGCGACCTACTCTAGGCAGTTGACGACTT GAATGCCCTAGCCGGTCTCAACCCGACGCCGCTCCACTTGCGCGACCAGCTGCGAGTATA GCGCGTTCACGCCTGGTTCTACTTCCTTAACGCGCGGAACCTCGCGCTCAATTACCTAGA CGCTGCGAGGACGCTATCGCGGGCTTGGAGCGCGCTCACGCCGTAAGAGTTGTCGAACCG GCTCGGGCGGACT

Inverse complement of above sequence:

CATAGATCGCTCTTCATTGACAAGGTTTTCCCCGGATGCCATCATTAGAAAACCTCTAGT AGATATAGAGTCAAGTTTTTTATAGGAAGCAACCATGATGCGGATCGGTGAACTGGGCAA GAAGGCAGATTGCTTGGTGCAGACCGTGCGCTTTTACGAGTCAGAAGGCTTGCTGCCCGA GCCTGCACGTAGCGAGGGCAACTTCAGGCTCTATGACGAAGTCCATTTGCAGCGCTTGCT GTTCATCCGCCGCTGCCGGGCGAAGGACATGACGCTGGATGAGATCCGTCAACTGCTGAA CTTACGGGATCGGCCAGAGTTGGGCTGCGGCGAGGTGAACGCGCTGGTCGACGCTCATAT CGCGCAAGTGCGGACCAAGATGAAGGAATTGCGCGCCTTGGAGCGCGAGTTAATGGATCT GCGACGCTCCTGCGATAGCGCCCGAACCTCGCGCGAGTGCGGCATTCTCAACAGCTTGGC CGAGCCCGCCTGA

Inverse Complement Sequence BLASTed:

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=93352797&dopt=GenBank&WebEnv=0cvy0r_s7OgtB31ICc6NXtgu1iJMjqrGLyFI4oYcQxj2kT9qtcQijdQrtTUUbUUmddm6suqFMcOWS9o%40255F25AC68A58C60_0055SID&WebEnvRq=1

gb|CP000352.1| Ralstonia metallidurans CH34, complete genome

Length=3928089

CP000352.1

Features in this part of subject sequence:

  transcriptional regulator, MerR family

  Heavy metal translocating P-type ATPase

Score =  911 bits (493),  Expect = 0.0

Identities = 493/493 (100%), Gaps = 0/493 (0%)

Strand=Plus/Minus

  /product="transcriptional regulator, MerR family"

                    /protein_id="ABF06988.1"

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=ABF06988.1

Indicates protein sequence is:

ORIGIN

1 msaqpstvty titdlarefd itprairfye dqgllapdre gpsgrrrvyn srertrlklt

61 lrgkrlgltl neireildly esprdtapql erflhllagh rgtlerqled lqaqlaeidq

121 herqcqalla aqhaknagnk tpta