Python - How do you replace a certain line in a text file with another string?

I believe it would be easier to keep track of the the mastery level using a dictionary stored in a pickle. It makes it very easy to handle program's state and is much faster to modify.
For a quick answer, I would load the pickle like this:

"""
We are loading the pickled mastery values by checking if the pickle file exists.
If it doesn't we define a default mastery level
"""
try:
    with open("mastery.p", "rb") as mastery_file:
        mastery = pickle.load(mastery_file)
except FileNotFoundError:
    mastery = {
        "1: Define isotope": 0,
        "2: Name the type of decay that occurs when, they have too many neutrons": 0,
        "3: Define Coulomb": 0,
    }

You can then use mastery as a regular dictionary and modify the values accordingly. When you are done, you can save the mastery state in the pickle again:

def save():
    with open("mastery.p", "wb") as m:
        pickle.dump(mastery, m)

Now for the entire program:

import pickle

"""
Here we defining the quesions and choices in a dictionary, but you could
load them from a normal text file, json file, or a pickle.
The questions are the keys to choices.
"""
q_and_a = {
    "1: Define isotope": [
        "[A]Atoms of the same element with same number of protons and a different number of neutrons",
        "[B]Atoms of the different element with same number of protons and a different number of neutrons",
        "[C]Atoms of the same element with same number of neutrons and a different number of protons",
        "[D]Atoms of the same element with same number of protons and a different number of electrons",
    ],
    "2: Name the type of decay that occurs when, they have too many neutrons": [
        "[A]Gamma Radiation",
        "[B]Alpha Decay",
        "[C]Beta Plus Decay",
        "[D]Beta Minus Decay",
    ],
    "3: Define Coulomb": [
        "[A]1 coulomb is the quantity of charge carried past a given point if a steady current of 1 amp flows for 1 second",
        "[B]1 coulomb is the quantity of charge carried past a given point if a steady voltage of 1 volts per 1 second",
        "[C]1 coulomb is the quantity of current carried past a given point if a steady voltage of 1 volts per 1 second",
        "[D]1 coulomb is the rate of flow of charge",
    ],
}

"""
The answers have been defined with the same scheme as the questions
Once again, you could load these from a file or add them as the last
item in the q_and_a and filter them out when giving the questions.
"""
answers = {
    "1: Define isotope": "a",
    "2: Name the type of decay that occurs when, they have too many neutrons": "b",
    "3: Define Coulomb": "a",
}

"""
We are loading the pickled mastery values by checking if the pickle file exists.
If it doesn't we define a default mastery level
"""
try:
    with open("mastery.p", "rb") as mastery_file:
        mastery = pickle.load(mastery_file)
except FileNotFoundError:
    mastery = {
        "1: Define isotope": 0,
        "2: Name the type of decay that occurs when, they have too many neutrons": 0,
        "3: Define Coulomb": 0,
    }

# Here, we are iterating over every question each session


def show_questions(q, choices):
    print(f"{q}:")  # Show the question
    print("\n".join(choices))  # Show the choices
    answer = input("You answer: ")  # wait for answer
    if answer.lower() != answers[q]:  # convert to lowercase (to match answerkey)
        print(f"wrong, answer is {answers[q]}")
        mastery[q] -= 1
        print(f"Your mastery decreased to {mastery[q]}\n")
        return False
    else:
        print("Correct!")
        mastery[q] += 1
        print(f"Your mastery increased to {mastery[q]}\n")
        return True


def save():
    with open("mastery.p", "wb") as m:
        pickle.dump(mastery, m)


def loop_all():
    """
    Loops through all qustions once
    """
    for q, choices in q_and_a.items():
        show_questions(q, choices)
        save()


def for_mastery(max_level=3):
    """
    Loops through each question till mastery is reached
    Mastery can be adjusted by passing it as an argument
    """
    for q, m in mastery.items():
        print(m)
        choices = q_and_a[q]
        while m < max_level:
            passed = show_questions(q, choices)
            if passed:
                m += 1
            else:
                m -= 1
            save()
        print("mastered!\n")


for_mastery()