Investigation of the link between genotype and phenotype

of alkaline phosphatase from different experimental

approaches in E.coli

Introduction

Phosphate is an indispensable nutrient for growth in all living

organisms. It is a major component not only in nucleic acids which

are basic units of DNA and RNA but also in ATP which is an energy

supplying molecule. However, a wide range of available phosphates

that occur in nature fail to be metabolised straightway by most of

the microorganisms such as Escherichia coli (abbr. E.coli) in this

experiment, unless they are first degraded to an intermediate,

inorganic phosphate (abbr. Pi). The primary reason regarding to this

incapability of immediate utilisation of phosphate is the permeability

of E.coli cell membrane.

Being a member of Gram-negative bacteria, E.coli is surrounded by

an outer and an inner membrane between which a periplasm

containing a thin layer of peptidoglycan is sandwiched. The outer

membrane acts as a molecular sieve with large permeability since

there is a presence of non-specific integral proteins called porins,

which forms channels through the outer membrane lipid bilayer and
allows most of the molecules entering the cell. Conversely, the inner

membrane is characterised as the main permeability obstacle where

phosphate-containing molecules are unable to penetrate and be

employed for particular use in cell. The problem is solved by having

an enzyme called alkaline phosphatase (abbr. APase) residing at

periplasm in which phosphate-containing molecule being blocked by

the inner membrane and incorporated by the outer membrane.

Once phosphate-containing molecules are in periplasm, they are

hydrolysed by APase which then results in liberation of Pi and a

yellow byproduct. These free Pi are then captured by specific

binding proteins and finally transported across the inner membrane

and genuinely utilised by the cell. However, in a special case where

point mutation has acted on the DNA sequence, the mutated APase

may not be able to cleave the phosphate and the resulting activity

may be profoundly affected. This is due to a functional APase is

synthesised through a cascading process which connotes a mutated

DNA sequence will result in a mutated mRNA transcript which is

then translated into a faulty amino acid sequence which in turns will

eventually codes for a faulty APase. Although point mutation can

have a deleterious effect on APase activity, it can also be

exceptionally conservative which means the amino acid introduced

by the mutation has similar properties to that of wild type owing to

the altered codon still codes for the same amino acid because of the

high degree of degeneracy of the genetic code. Thus, an APase

originated from a conservative mutation will still have a similar

biochemical activity as that of wild type. Likewise, an APase

originated from a non-conservative mutation will have an absolutely

opposite response in activity to that of wild type.

As a result that manufacture of APase is energy costly, the

subsequent issue will be illustrating how E.coli cells sense that

synthesis of APase is unnecessary and not urgent. The answer to

this is the regulation of phosphate regulon (abbr. pho regulon) which

encompasses the expression of APase's structural gene, Pho A.

In wild type strain of E.coli, Pho A gene coding for APase is

expressed and APase is synthesised in large amounts only when Pi

becomes limiting in the medium whereas APase is repressed when

growth occurs in the presence of excess amount of Pi in the

medium. However, in a special scenario where a constitutive

mutation of pho regulon occurs, whose circumstance is defined as

constant expression of a gene product regardless of environmental

conditions, the repression is not inhibited in spite of the presence of

Pi. There are two different classes of constitutive mutation. One is

operator mutant and the other is repressor mutant. Their incurred

mutation outcomes can be speculated by the names. Namely, an

operator mutant is a mutation taking place at the operator

sequence that affects the binding action of a repressor which in

turns causes Pho A gene to be continuously transcribed and

translated to APase regardless of presence of Pi. Similarly, a

repressor mutant is itself mutated and so fails to bind to the

operator resulting in the same outcome as in operator mutant which

is APase is synthesised endlessly with Pi existing medium.

Therefore, based on the detailed knowledge provided above, the

hypothesis in the third week experiment would be a yellow product

is an indication of APase activity and a wild type strain of E.coli

ought to have an opposite responses in Pi and without Pi

environment. Additionally, if a there is a constitutive mutant, two

positive responses should be observed in both Pi or no Pi instances.

Aim
To illustrate the relationship between genotype and phenotype by

using mutants to link changes in DNA sequence to phenotypic

changes and to deduce the underlying genetic processes.

Materials and methods

Results

week ① experiment

Figure 1.) Rate of alkaline phosphatase activity in four E.coli

strains

Figure 2.) Rate of alkaline phosphatase activity in four E.coli

strains

Two graphs from the first week experiment illustrate the rate of

APase activity in four E.coli strains from different aspects on xy-axes

but the same conclusion will be drawn. In figure one, the line of wild

type is noticeably steeper than mutant #2 whereas both mutant#1

and negative control are completely flat on the x-axis. The

observation from those lines suggest that wild type has the highest

rate of APase activity while mutant#1 and negativity control has nil

APase activity and the APase activity in mutant#2 is in between.

From another point of view, figure two further evaluates the rate of

APase activity with an actual value derived from the slope which is

the gradient of the line in figure one. The values utterly echo the

suggestion stated in figure one with the wild type being the highest

rate of 0.0428 (A420/1010cells/min) in APase activity, mutant#1 and

negative control having no activity and mutant#2 lying between the

two extremes with the rate of 0.0288 (A420/1010cells/min) in APase

activity. However, all the results are referred to the class result

because of mis-pietting of the solution.

Week ② experiment

Figure 3.) Gel electrophoresis photo

 Figure 4.) DNA size against migration distance

In figure four, the trend from the best fit of size marker deduces

DNA fragment sizes in three E.coli strains Each of them will have

two settings which are possessing a restriction enzyme and no

restriction enzyme as a control respectively. In all the undigested

strains and digested wild type of E.coli, their measured 28mm

migration distance from the gel photo correspond to log(3.060) size

of DNA fragment from the trend. Additionally, digested mutant#1

has two daughter DNA fragments with size of log(2.600) and

log(2.825) whereas mutant#2 has presumably two daughter DNA

fragments of the same size which is log(2.750).

Table 1.)

Sample Migration Size (bp)

distance(mm)

Wild type-undigested 28.0 1148

M1-undigested 28.0 1148

M2 -undigested 28.0 1148

Wild type-digested 28.0 1148

 31.0 668

35.5 398

M2-digested 33.0 562

Table one simply solves the DNA size which is in logarithm form in

terms of a whole number against their migration distance on the

gel photo. Again, from the table, all the undigested strains and

digested wild type of E.coli generally have 1148 bp of DNA size

while mutant#1 has two DNA sizes of 668bp and 398bp

respectively and mutant#2 has two DNA sizes of the same length

which is 562bp.

Figure 5.) PCR diagram

M1 -

digested
M2-digested

Figure five establishes a more descriptive way to present the cut

daughter DNA fragments in mutant#1 and #2. From both

diagrams, DNA in mutant#1 is digested into two different sizes

where the shaded areas exemplify a typical ritual that a restriction

enzyme will follow that is sever the DNA in a zigzagged pattern.

Similarly, DNA in mutant#2 is digested into two and once again

the shaded areas indicate that the restriction enzyme prefers to

cut the DNA in a zigzagged pattern yet unlike mutant#1,

mutant#2 ends up halving the DNA into two equal sizes.

Week ③ experiment

Table 2.) Indication of APase activity with the presence and

absence of Pi in four strains of E.coli

 Alkaline phosphatase activity

+Pi -Pi

Wild type

Mutant #4 　

Mutant #5

Mutant #6

Table two summarises the activity of APase in four strains of E.coli.

A positive response accompanied by a yellow colour in the

Eppendorf tubes implies APase activity and vice versa, without the

yellow colour implies no APase activity and is a negative response.

From the table, a positive response lies in strain of wild type with

the absence of Pi, of mutant#4 with both presence and absence of

Pi. The rest of the scenarios are all negative responses. However,
the results in wild type was retrieved from class results. Two

plausible reasons for this error may be either there exists

contamination in the tube or there is no phosphate in the tube.

Discussion

As the aim of the experiment is to illustrate the relationship

between genotype and phenotype by using mutants to link changes

in DNA sequence to phenotypic changes, experiment one can be

discussed in conjunction with experiment two to demonstrate this.

All data from experiment one and two in results section are

consistently supported by the theory and the hypotheses in the

introduction. For example, since a wild type strain of E.coli has no

mutation taking place, a functional APase is synthesised and will

reasonably display the activity. This is proven by using the

spectrophotometer to obtain the highest rate of 0.0428

(A420/1010cells/min) APase activity among all strains. Moreover, an

action of restriction enzyme can be taken on the DNA sequence only

when there is a point mutation that can potentially introduce or

remove restriction enzyme sites. Without any mutation, there won't

exist any restriction enzyme site for a restriction enzyme to

recognise and perform cutting in wild type. Therefore, the gel photo
depicts no daughter DNA fragments and the distance travelled by

parent DNA fragment in digested wild type is equivalent to that in

undigested wild type.

In comparison with non-mutated E.coli wild type, a different

phenotypic response of APase activity ought to be observed in

mutants. Mutant#1 follows this logic and exhibits no APase activity

via spectrophotometer. The theory explained earlier on in the

introduction that a defective APase is caused by an altered DNA

sequence affecting the following cascading steps of protein

synthesis is further demonstrated in the result of mutant#1 at a

genetic level. From the gel photo, mutant#1 is found to be digested

into two daughter DNA fragments whose sum (1066bp) is relatively

close to the distance of digested wild type (1148bp) hence the idea

that the restriction enzyme sites are inserted by point mutation for a

restriction enzyme to operate is enhanced.

On the contrary, although mutant#2 has been digested by a

restriction enzyme into two daughter DNA fragments of the same

size where the doubled amount (1124bp) is also comparatively close

to that in digested wild type (1148bp), its APase phenotypic activity

is extremely dissimilar to that in mutant#1. APase in mutant#2 is

unexpectedly active at a rate of 0.0288 (A420/1010cells/min) instead

of zero activity. This shows that a point mutation can develop into

different outcomes. In this case, mutant#2 is regarded as a

conservative mutant whereas mutant#1 is deemed to be a non-

conservative one. Again according to the theory in introduction, a

conservative mutation means the amino acid introduced has similar

properties to that of wild type owing to the altered codon still codes

for the same amino acid because of the high degree of degeneracy

of the genetic code, the active site of APase in mutant#2 has not

been disrupted and thus can still function its phosphate metabolism.

Similarly, the active site of APase in mutatn#1 has been disrupted

due to the incorrect folding of altered amino acid sequence in 3-

dimensional space and consequently it can no longer bind to a

phosphate substrate and is said to be non-conservative. However,

through gel electrophoresis, it can be deducted that the type of

point mutation in mutant#1 is more likely to be a deletion mutation.

The deduction can be accounted for the deficient genetic

information that is encoded by the shorter daughter DNA fragment

(398bp) in digested mutant#1. Compare this 398bp DNA size in

mutant#1 with 562bp in mutant#2 whose APase displays normal

activity, the DNA length of mutant#1 is too short to have sufficient

genetic information needed for coding a functional APase. Thus,

mutant#1 is more susceptible to a deletion mutation which results

in pre-mature translation termination of an amino sequence.

The aim of week three experiment is to propose an underlying

genetic model deduced from phenotypic changes of several E.coli

mutants in phosphate acquisition. The hypothesis corresponds to

the data shown in the results which is a wild type has an opposite

response and mutant#4 is considered to have constitutive mutation

as it displays positive responses in both Pi-sufficient and Pi-deficient

media. The opposite responses in wild type of E.coli is because in an

abundant Pi environment, the cell will save the energy to transcribe

and translate the phoA gene coding for APase and freely obtain Pi

from the environment directly. Conversely, in a Pi shortage

environment and the cell is starved for Pi, phoA gene is expressed at

high levels and APase production is induced. Thus, Pi can possibly

be a co-repressor that activates repressor to bind to the operator

and switch off pho regulon. However, the case where mutant#4

shows both positive responses is due to a mutation in the regulator

gene where a repressor is formed and released codes for a mutant

repressor that has a conformational change. This results in even if Pi

is present, it fails to play its role as a co-repressor, encouraging

repressor to bind to the operator and stop the expression of phoA

gene in to APase, since the shape of repressor is now different.

Based on the gene mapping diagram, as a result that the positions

of mutation in mutant#4 and mutant#5 are close to each other,

mutant#5 can be speculated to be a repressor mutant as well.

Unlike repressor mutant#4, the mutated repressor in mutant#5 is

assumed to always bind on the operator so that phoA gene coding

for APase will never get expressed. This speculation is supported by

the negative response in the absence of Pi. Likewise, mutant#6 also

presents a negative response in Pi-deficient medium but is

suggested to be an operator mutant due to the position of mutation

is at the upstream of transcriptional phoA gene. The mutation

occurs in the operator sequence of the pho regulon and significantly

causes a repressor to bind more tightly to it and therefore blocks the

transcription of phoA gene which in turns inhibits itself translated

into APase.

Conclusion