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SBB 3033: PRINCIPLES IN MICROBIOLOGY CASE STUDY: GENE ANALYSIS NAME Ahmad Kamil Bin Mohd Jamaai Mohamad Zarif Muaz Bin Roslan Mohd Hafiz Bin Salleh Ridwan Bin Shamsudin MATRIK NO D20101037 521 D20101037 494 D20101037 433 D20101037 472

Submitted to : Dr. Wong Chee Fah

CASE STUDY: ANALYSIS OF GENE SEQUENCE (AY738723) a. Identify type and source of the gene. How? (Please include print screen of the finding)
Organic solvent-tolerant lipase gene. The information can be obtained by entering the number of locus into http://www.ncbi.nlm.nih.gov/. In this case the locus number is AY738723. We must to make sure that we choose the nucleotide in the search category. The source is from Pseudomonas aeruginosa.

Figure 1

b. Provide brief introduction of the gene. (Search for related literature, preferably journals, regarding the gene)

Pseudomonas sp. strain S5 was locally isolated from soil. This strain has been identified as a BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene) degrader. This strain generates a lipase which is stable in the presence of organic solvents including nhexane, cyclohexane, toluene, and 1-octanol Pseudomonas sp. strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. There are reported that the isolation of organic solvent-tolerant Pseudomonas strains which most effectively secreted lipolytic enzyme. These strains were designated as strains LST-03 and S12.There are many researchers have found that among lipases of various origin (animal, plant, and microorganism), those from bacteria, particularly from Pseudomonas species, evidence the highest degrees of versatility, reactivity, and stability in the catalysis of reactions in organic phase. Reactions catalyzed by lipases are normally conducted in organic aqueous two-phase media, which are favorable because they render the separation of enzyme from substrates or products fairly easy.

c. Translate the nucleotide sequence. (Please include print screen of the finding)

Figure 2

d. Identify the following region. What is the function for each of the region?

A) Promoter There is no promoter for this sequences.

Figure 3

Function of promoter : A region of DNA that initiates transcription of a particular gene. Promoter also to signal the RNA polymerase where to start transcribing the DNA.

B) Shine Dargano

Figure 4

Shine dalgarno sequence is at tcaggaga at 291 of nucleotide sequence. Function of Shine Dalgarno is to help recruit the ribosome to the mRNA to initiate protein synthesis by aligning it with the start codon.

C) Signal Peptide

Figure 5

The signal peptide These data presented the values of the C, S and Y scores for the signal peptide and cleavage site for S5 lipase. The sharp peak indicated the C score at position

36 between amino acids VHA-AT. This peak corresponded to the change in the value of the S-score from a high to a low value. The cleavage site is located at position 3537, indicated by the maximal Y-score. D) Propeptide

Figure 6

Figure 7

Figure 8

Propeptide is at sequence 165 translation protein and at sequence 196.

The function of propeptide is a protein precursor is an inactive protein or peptide that can be turned into an active form by posttranslational modification. Pro-peptide also part of a protein that will cleaved during maturation or activation.

E) Mature Protein

Figure 9

Mature protein sequences from 197 of protein translation until the stop codon which is 448 of protein translation. Function of mature protein is protein that released from the proprotein that undergoes cleavage by a serine endoprotease within cellular membranes, such as the ER or Golgi. F) Start Codon

Figure 10

Start codon at sequence 19, 114 and 439 at the sequence of protein translation. Function of start codon is the first codon of a messenger RNA (mRNA) transcript translated by a ribosome. The start codon always codes for methionine in eukaryotes and a modified Met (fMet) in prokaryotes. The most common start codon is AUG.

G) Stop Codon

Figure 11

The sequence for stop codon is 1624 for nucleotide sequence. The function of stop codon is a nucleotide triplet within messenger RNA that signals a termination of translation. Proteins are based on polypeptides, which are unique sequences of amino acids. Most codons in messenger RNA (from DNA) correspond to the addition of an amino acid to a growing polypeptide chain, which may ultimately become a protein. Stop codons signal the termination of this process by binding release factors, which cause the ribosomal subunits to disassociate, releasing the amino acid chain.

e. In your own words (flow chart), provide the methodology for cloning and expression of the gene based on the supplied papers.
Methodology for cloning and expression of the gene AY738723

Figure 12

f. Based on our discussion during the lecture and information from the relevant papers, how do you know if

a. the gene is transcripted and translated into an active enzyme. In bacteria, these two steps are closely linked. Transcription starts when RNA polymerase binds to the promoter region of the gene, and proceeds to copy the DNA sequence into mRNA. As the 5' end of the transcript ( i.e., the mRNA) is made, ribosomes immediately attach to it, and start translation, even before the entire message is synthesized. The ribosomes move along the message, translating the mRNA, as it is being made. Behind the ribosomes, the 5' end of the message is rapidly degraded. b. the gene is successfully transcripted but not translated. To initiate transcription, the polymerase first recognizes and binds a promoter region of the gene. Thus a major mechanism of gene regulation is the blocking or sequestering of the promoter region, either by tight binding by repressor molecules that physically block the polymerase, or by organizing the DNA so that the promoter region is not accessible. We can determined that the gene is not translated if mutation occur. c. the gene is transcripted and translated, but not into active enzyme (inclusion bodies). Foreign gene product may be toxic to a bacteria. Allowing the gene to be constantly transcribed and translated could result in killing bacterial cells. Sometimes, if the protein is not toxic to the bacteria, production of large quantities of the protein could lead to the formation of inclusion bodies which are structure in cell that contain aggregate of insoluble protein.

g. You are required to express a gene encoding -35 and -10 boxes, Shine-Dalgano, signal peptide and mature protein from a cloning vector. Is that possible? Why?
Yes it is possible. In prokaryotes, the promoter consist of 2 short sequences at -10 and -35 positions. Ribosome can only find the correct start codon by first binding the Shine-Dalgarno sequence, so the vector should also have this sequence positioned just before the desired start codon. Eukaryotic gene have regulatory signals like enhancers and TATA boxes that are not recognized by bacteria and lack the -10 and -35 sequences that RNA polymerase needs to start transcription. In addition, eukaryotic ribosomes find the correct AUG codon by scanning from the cap to the first AUG, while bacteria rely on a Shine-Dalgarno sequence in the mRNA. These bacterial regulatory signals are provided by the expression vector. If the plasmid that do not have these signal are used, it is very unlikely that the bacteria would be able to transcribe and translate our gene.