Virtual Lab Report

Name of the Simulation

Labster: Gene Expression
This lab report is for you to reflect on what you completed and learned in this simulation,
and to practice your written scientific communication skills.

Sections

1. Describe the overall objective and make a hypothesis

2. Introduce relevant background knowledge on this topic
3. Summarize the steps taken in the simulation
4. Explain any obtained results
5. Discuss the conclusions and implications

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 1. Describe the overall objective and make a hypothesis.
What is the overall purpose of the experiments or activities? Make a hypothesis if applicable.

Hint: The purpose is often stated in the welcome message of the simulation.

The purpose of the lab is to get familiarized with sequencing DNA and estimate levels of a gene expression of a specific gene. There

were no hypothesis to be formed in this simulation.

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 2. Introduce relevant background knowledge on this topic.
What have you learned in class or researched on your own that would help prepare for this
simulation?

Hint: You can review the “THEORY” section in the simulation or at https://theory.labster.com/
if you need help.

Next Generation Sequencing can be used to sequence transcript of specific genes and evaluate their relative expression profiles, this

way we can compare genes of subjects with controls to see which genes contribute to diseases. NGS can also be used for other things

like whole genome sequencing. mRNA always start with 5' UTR to mark the start and at the 3' end we have A tails which marks the

end of the mRNA. Taq polymerase can only "synthesize" DNA, but not from mRNA. The cDNA is made of mRNA and a reverse

transcriptase. Highly expressive genes will see many cDNA molecules because cDNA and mRNA are a 1:1 ratio. Our cDNA need

adenine bases to be read. Taq polymerase can attach adenine overhangs onto the cDNA. Now the adapter can add their 3' Thymine

ends, enzymes help with this in the simulation. All reactants from before other than the DNA are removed with magnetic beads.

Primers are then added, as well as polymerase, and the DNA is copied 10-12 times. Reads can tell us if the gene is expressed a lot or

not, the more it is expressed, the more "reads" there are. Leptin found a lot more read in the healthy pig compared the unhealthy pig,

leptin controls appetite, which might be why the pig with diabetes eats more. qPCR can be used to see gene expression, it counts the

number of fluorescence after each cycle. Reference genes are used to see the LEQ gene expression, we need to find genes that are

stable in samples to use it as a reference. However, we have to be aware of cross contamination because it can effect the result very

much. We also saw the simulator use 2 samples for each, that being the water control, GAPDH qPCR mix and hte LEQ qPCR mix.

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Both lean and obese pigs had detectable PCR products at cycle 22(LEP).

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 3. Summarize the steps taken in the simulation.
Explain each step you completed including the equipment and techniques you used.

Hint: You can use the “MISSION” tab in the LabPad as inspiration.

We first obtained the mRNA from two pigs, one obese and one not, we want to compare their DNA using NGC and link certain genes

to mutation. We needed to make cDNA from mRNA by using reverse transcriptase, then we converted cDNA into actual DNA strands

using taq polymerase and adaptors to add the A and T overhangs, and adaptor primers, polymerase then copies the DNA under high

temperature. After we have plenty of DNA sample, we need to generate clusters, which is done by the NGS machine, allowing us to

sequence the DNA. The flow cell surface is covered with two types of attached small DNA molecules that are complementary to the

two adaptor sequences, which binds to the single stranded DNA samples, the opposite end binds to another small DNA molecule,

creating a bridge shape. It is then replicated using the adaptor sequence as the primer and polymerase. When the bridge is opened, we

now have 2 DNA clusters with strands of complementary DNA in a "standing" shape. One of the two

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clusters are washed away, leaving us with only one cluster of identical DNA in all the two cluster pairs. Once the base is attached, we

can see the polymerase attaching each of the nucleotides, allowing us to photograph and sequence it. Next, we analyze the data. We

first discard the low quality data and then trim the sequence of the adaptors. We can now see the reads of the genes. After mixing

cDNA with the LEP master mix with all the enzymes we need. We need to find a reference gene so we can have relativity to the

amount of gene expression the LEP gene is expressing. The PCR product should be doubled each cycle if the efficiency is 100%.

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 4. Describe any obtained results.
Explain any obtained results. Were these results expected or unexpected?

Hint: You can use the “MEDIA” tab in the Lab Pad to find relevant images from the simulation.
You can also take screenshots while you are playing the simulation.

There were no data to be collected in this data, everything that is shown is taught right way since the purpose of this lab wasn't to

solve a problem. The only numerical data was the Ct cycles in where GAPDH and LEP are detectable, it is 24 and 22 in obese pigs

respectfully, and 20 and 22 lean pigs. Since there are more 4 cycles, there are 16 more times GAPDH, so the lean pig had 16 times

less LEQ levels because it is proportionate. We can determine that LEP indeed cause obesity, at least in pigs.

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 5. Discuss the conclusions and implications.
How do your results relate back to the original purpose and your hypothesis? Were there any
systematic sources of error that could have affected the results? What did you learn? What is
the importance of these findings and how can you apply them to other real world situations?

Overall the lab was very educational and the longest one I've done. Since there were no hypothesis, there was nothing for me to

prove. We have known for a long time now that genetics can lead to obesity, so that wasn't an appropriate hypothesis. However, we

were able to find the gene that was causing obesity in the pig by first using DNA sequencing to locate the gene, and then testing the

expression to make sure that it is the one making the difference in the pigs' health. There were no sources of error other than the fact

that a lot of times you could just leave the test tubes in the machines and it would reset for you. This is discussed in the experiment to

be very detrimental because it can lead to more frequent cross-contamination, effecting the results. I have learned all the steps to get

mRNA into DNA, and how sequencing it works. I have also learned how to confirm the data using qPCR to quantify and amplify the

result. There aren't much real world situation but this has very much given me a heads up to higher level biology, which is always

exiting.

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