Newcastle University Drylab/2 June 2008

From 2008.igem.org

Bugbuster-logo-red.png
Ncl uni logo.jpg


Newcastle University

GOLD MEDAL WINNER 2008

Home Team Original Aims Software Modelling Proof of Concept Brick Wet Lab Conclusions


Home >> Dry Lab >> Dry Lab Journal

May
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
June
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30
July
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31
August
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

2 June 2008

Mark

Today began implementing the exercises set in the Java tutorial into my EA. I created two classes called Node and Promoter, where I created a number of promoter objects to be associated with each node. This has now replaced the double arrays that I started off with in my program. Each promoter object has a name prefixes with P, followed by a number (from count). I have defined 14 different promoters, and for each node 5 of these ate selected randomly and input into a subArray which is then added to the Promoter array in the Promoter class. This is carried out for each node in the three layers in the network, the input/hidden/ and output layers. A double threshold value has also been stated, which in this case has been set to ten. Only when the promoter threshold values in the five promoters add up to ten will the fitness be accepted. Untill this point the EA will mutate the network. This mutate method is what I will begin writing tonight and tomorrow.

Have also spent some time today investigating the LisRK and agr two component systems in Listeria monocytogenes. Delved deep into the literature and also carried out a number of Bioinformatics analyses using UniProt, pfam (although it was down for most of the day), Kegg and Blast. The conclusions that I have drawn from this are that the LisRK system is very unique (in terms of identity), but we would be sensing the unknown, as an A-I has not yet been identified for this organism. In the arg system, I have discovered that again there is only 100% identity for the agrC sensor kinase, but similarity is quite high for one other Listeria species. These findings need to be discussed in full in the meeting on thursday before going any further with this organism. The findings in terms of papers and Blast results are shown below for Listeris monocytogenes:

This evening continuing in the same vein with Staphylococcus aureus.


Megan

Protein analysis using bioinformatic tools on the vnc operon. Firstly I found the EMBL record of the genomic region that contains the vnc system. This contains the vncS, vncR, coding region for the peptide that is sensed. These are parts that I alreaady knew would be involved in the two component system. There are also a number of hypothetical proteins, conserved regions and transporters. Fisrtly the 3 aforementioned parts will be analysed then I will move on to assess the other proteins, their role and whether they are required within the system to be used.

Gene name Product BLAST information (other similar species) Protein domain Domain funtion
CysS Cysteinyl tRNA synthetase 79% identity to S.gordonii and other Streptococcus species Cysteinyl-tRNA synthetase Aminoacylation catalysis
Rossman-like α,β,α sandwich fold Tyrosyl-Transfer RNA Synthetase
Cysteinyl-tRNA synthetase class DALR DALR a family found in this protein
Cysteinyl-tRNA synthetase class la N-terminal Attachment of aa to tRNA. And catalytic domain
Cysteinyl-tRNA synthetase class la C terminal
spr0520 Conserved hypothetical protein Similar to other strep. Ribonuclease 3 Processes RNA
RIBOc Processing of RNA
Predicted inactive ribonuclease III homologue Endoribonuclease
spr0521 Conserved hypothetical protein Similar to other strep. NO HITS
spr0522 Hypothetical protein Some small regions have similarity to other organisms NO HITS
Transposase H-truncation Transposase, uncharacterised, truncation Similarity to S. agalictiae, 95% coverage with 78% identity Group II introns maturase-specific Binding of introns RNA in reverse transcription and splicing
vex1 ABC transporter membrane-spanning permease-Pep export Not really seen in other organisms Unknown DUF214 permease predicted Transport of lipids across inner membrane
Signal peptide
Transmembrane region
vex2 ABC transporter ATP-binding protein-Pep export Not really seen in other organisms Transporter regions ATP binding transporter. Translocation of compounds across membranes
AAA ATPase core Energy dependant unfolding of macromolecules
Binding cassete
unintegrated P-loop containing nucleotide triphosphate hydrolases
vex3 ABC transporter membrane-spanning permease-Pep export Not really seen in other organisms DUF214, permase predicted Transport of lipids across inner membrane
Transmembrane regions Predicted transmembrane spanning regions
pep27 A secreted peptide sensed by vnc Corresponds to pep27 NO HITS
vncR VncR, response regulator Top hits for S.pneumoniae then only 32% coverage Response regulator
Sensory transduction phosphorelation regulator DNA-binding. Recieves signal in two component system.
Transcription regulatory protein Involved in DNA binding
Sensor- Histidine kinase Sensor
vncS VncS, histidine kinase Other organisms only 2% coverage ATP binding region Chaperone. Structurally related ATPase domains of histidine kinase
HAMP region Transmembrane proteins, part of 2-C pathway
Hist. kinase A – N terminal Dimerisation and phosphoacceptor domain
Signal peptide and transmembrane regions Sensor for histidine kinase also a sig pep and transmemb. Regions
fba Fructose-bisphosphate aldolase Found in other species also, especially pyogenes Ketose-bisphosphate aldolase class II Catalyses reversible cleavage. Involved in a number of pathways but doesn’t seem to be involved in 2-C system
Aldolase type. TIM barrel Catalyses reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate
Fructose-1,6-bisphosphate aldolase, class II

This analysis highlights inforamtion on the proteins that we were expecting to use. However I have not uncovered the repressor that is thought to be present. The case may be that this is not contained in this section of the genome. Further work is required to try to find this as it would be a problem if we are unable to use this two-component system.

Attempted to start the Java exercise that has been set by Morgan for my individual project. Didn't go very well, ended up a big java mess, very confusing to get my head round- probably should have tackled this during the daytime when Morgan was around.


Morgan

Nina