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Operon Fusions Lab Report

Specialized transuding phage Mud1(lac, bla)containing a gene encoding for ampicillin resistance and the lac structural genes lacking the promoter was constructed by recombinant DNA and used to transform MC4100 strain of E. coli. Transposition of the Mud1 (lac, bla) phage to chromosomal sequences resulted in lac expression being controlled by chromosomal promoter of the bacteria and mutation of the arabinose genes if the insertion was in the arabinose operon (Casadaban and Cohen, 1979. )

Selection for transformed MC4100 in general was done by selection for ampicillin resistance on media containing ampicillin. Only transformed bacteria survived in the presence of ampicillin. Selection for arabinose resistance was done on media containing arabinose. The patching of the ara-lac fusions on the appropriate media gave information on the mode of regulation of lac genes and the specific point of insertion of the phage bearing the lac gene within the arabinose operon.

Fraction of ara::Mud1 insertions lysogens were determined by plating ara-lac fusions on different plates and incubated. The phenotypes of the colonies selected were determined; the information was utilized in determination of the specific point of insertion of the phage within the arabinose operon. Introduction In this experiment a transducing phage Mud1 carrying bla (for ampicillin resistance) and lac structural genes without a promoter (reporter genes) was used to transform MC4100 bacterial strain and selection for ara-lac operon fusions carried out on appropriate media.

The main purpose of the experiment was to select for transformed MC4100 bacterial strain in two step procedure, first select for transformed MC4100 in general by selecting for ampicillin resistance and then select those bacteria infectected by the phage in the arabinose operon in specific by selecting for arabinose resistance When these Mu derivatives (Mud1) are inserted in a gene in the correct orientation, the lac genes are expressed from the promoter of the mutated gene. Hence, expression of the lac operon has a direct proportionality to expression of the mutated gene.

Given that expression of the lacZ gene is easily detectable on indicator plates. Operon fusions put the transcription of a reporter gene under the control of the promoter of a target gene but the translation of the reporter gene and target gene are not dependent (Beatriz, Olson, and Casadaban 1984). if the phage carrying lac genes is inserted behind an active promoter in the forward (ZYA) direction, the expression of the lac genes under the control of target gene promoter in this case, araC or arabinose structural gene promoter was demonstrated by plating on an indicator media.

The media turned red due to acid formation which was a clear indicator that the bacteria did utilize lactose for as a carbon source. The results obtained give a clue on how the lac genes are regulated. The expression of arabinose operon structural genes araBAD is dependent on the presence of both the substrate L-arabinose and the protein product of the araC gene, which is apositive regulator of the operon. Any mutation in araA, araB or araC could be detected by selection on the arabinose media since the bacteria cannot utilize L-arabinose in the absence of the enzyme products of these genes.

The patching of the ara-lac fusions on the appropriate media give information on the mode of regulation of lac genes and the specific point of insertion of the phage bearing the lac gene within the arabinose operon. Discussion The experiment is divided into different parts each with a specific objective hence the discussion will critically and in detail analyze each part as an entity and then as part of the whole experiment.

The first part is dedicated to preparation of Mud1 lysate from the strainMAL103 the purpose of incubation at 42 degree centigrade was to lyse the bacteria and hence release the plasmids (Mud1) required later in the experiment for the transformation of the MC4100 strain. This was followed with preparation of titer lysate-for plaque forming units to obtain a dilution that was to produce between 30 to 300 plaques, which in this was a dilution of ten to power negative six which produced about one hundred and forty-one plaques after incubating the MC4100 with Mud1 at 30 degrees centigrade.

There were no colonies in the phage control plate since the MAL103 was lysed at 42 degrees centigrade temperature. This was followed with the determination of the frequency per plaque forming unit at which Mud1 formed lysogens i. e. the fraction of plaque forming units transformed by the phage. This was achieved through selection for Ampicillin resistance gene, on lactose MacConkey+ampicillin plate, since only the transformed bacteria would survive in the presence of Ampicillin due the presence of beta- lactamase gene in their genome which confers Ampicillin resistance.

There were both red and white colonies formed. The red colonies were due insertion of Mud1 behind an active promoter in the forward orientation (ZYA). This resulted in transcription of the lac operon structural genes and hence metabolism of lactose, followed with acid production which makes their colonies appear red. The white colonies were as a result of transformed bacteria utilizing peptones as a carbon source in the media hence no acid production.

The number of white colonies was one hundred and forty colonies while red colonies was thirteen in the dilution lysate of ten to power to negative which was taken as the optimum dilution in this case. The white colonies were due to Mud1 insertion behind active promoter in the reverse orientation. Selection for arabinose resistance was achieved by plating of transformed MC4100 on araMac+amp plates and one control without ampicillin to cater for spontaneous mutations frequency.

Fraction of ara::Mud1 insertions lysogens were determined by plating ara-lac fusions on different plates and incubated. The phenotypes of the colonies selected were determined; the information was utilized in determination of the kind of insertion. In this experiment two patches had white color patch in all the three media combination i. e. they were white in arabinose MacConkey, lactose MacConkey, arabinose+lactose MacConkey.

this is due to insertion of the phage at either araA or araB or araC in the reverse orientation hence inhibit arabinose operon due to deactivation of araC whose product is essential for the activation of ara operon structural genes in the presence of arabinose. Due to the reverse orientation of the lac structural genes they were not transcribed hence the bacteria was unable to utilize lactose as carbon source. In both cases their was no acid formation as the bacteria utilized peptones as carbon source instead of the sugars hence the colonies remained white.

Some patches had white color patches in both, lactose MacConkey and, arabinose MacConkey but red color in, lactose+arabinose MacConkey media. This was due to insertion of the phage in the araA or araB in the forward orientation, this blocks arabinose operon hence the bacteria is unable to utilize arabinose as a carbon source. The inability of the bacteria to utilize lactose was due to fact that the ara structural gene promoter is inactive in the absence of arabinose hence the lac structural genes were only transcribed in the presence of arabinose.

This resulted in the utilization of lactose making the patch color red only in the presence of the two sugars. In the patches with red patch color only in the presence of lactose is due to insertion of the phage in the araC with lac structural genes in the forward orientation. This resulted in the complete inhibition of the ara operon due to deactivation of the araC gene whose protein is essential for its activation in the presence of arabinose.

since the lac structural genes were inserted behind an active araC promoter in the forward orientation they were transcribed hence the bacteria was able to utilize lactose as a carbon source resulting in acid production making the colonies red. Reference List: Beatriz, A. , P. Olson, and M. Casadaban. 1984. ‘Plasmid insertion mutagenesis and lac gene fusion with mini-Mu bacteriophage transposons’. J. Bacteriol. l58: 488-495 Casadaban and Cohen, 1979. ‘Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage’. Washington D. C

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