BiochemAdventures

You have finished your undergraduate degree and discovered that you love [[biochemistry|Biochemists are awesome!]]. Now you are questioning what you want to do with your life. Because the next decision will greatly impact your life, you decide to try out biochemistry and land a job as a technician in a biochemistry lab. You are [happy. ☺]<hook| <img src="happier.png" width="200" height="200"> (Click: ?hook)[In your first week you are pleased to your new lab is fully equipped and stocked with anything you might need. Your supervisor, called a PI (principal investigator), outlines what they need from you. The group is trying to biochemically analyze a new enzyme; other than the DNA sequence and organism it expresses from, essentially nothing is known. [[Welcome to the real world!|RealWorld]]]

<img src="happybiochem.jpeg" width="720"> Happy undergraduates in the Bourne Lab, Spring 2015

You are on a deadline, and your results matter! • You will need to have the means to get protein in some form • You will need to have this protein purified by > 95% • You will need to be able to demonstrate some form of enzyme activity and maybe even inhibition To simplify this exercise, there will be four - five basic options for each section. Your job will be to interpret the task, the possible answers, and decide which will yield the best outcome in the shortest time frame. Some options are better then others. This means that sometimes all answers are essentially OK, but some are better than others, and at least one will be a great answer. Some answers will require feedback and re-assessing. [[Let's begin!|start]]

The enzyme has been named ParT, expressed from the parT gene found in E. coli strain 3753. Your first assignment is to come up with a strategy of obtaining this enzyme; your options are listed below: (A) [[Write to the investigator|1a]] that discovered the enzyme and ask if they could send you something to start from (B) Find this strain of E. coli, purify the genomic DNA and try to [[amplify the gene by PCR|1b]] (C) Find this strain of E. coli and [[try to purify the ParT enzyme directly from the bacteria|1c]] (D) Use a commercial source and ask them to [[synthesize the gene|1e]] (E) You find a [[commercial source that sells a protein|1d]] with the same sequence

After 1 week there is no response. Do you: A) [[Resend the request|1a a]] B) [[Contact someone else|1a b]] C) [[Choose again|start]]

Good choice! You easily find the correct strain and purify the genomic DNA. You also design primers to the gene, but just before ordering end up confusing yourself a bit. Which of the proposed primer pairs will successfully amplify the gene? Gene sequence 5' - ATG CTG GGC TGG CAT ................GTT ATG GTC AAA TGA - 3' 3' - TAC GAC CCG ACC GTA ................CAA TAC CAG TTT ACT - 5' [[A)|1b nope]] Forward: 5' - TCA TTT CTG ATG TTG Reverse: 5' - ATG CTG GGC TGG CAT [[B)|1b nope2]] Forward: 5' - AGT AAA CTG GTA TTG Reverse: 5' - TAC GAC CCG ACC GTA [[C)|1b yes]] Forward: 5' - ATG CTG GGC TGG CAT Reverse: 5' - TCA TTT GAC CAT AAC <img src="happy.png" width="200" height="200">

You have found this strain of E. coli and tried to purify the ParT enzyme directly from the bacteria. However, you find that the enzyme concentration in the cells is very low, too low for our needs. A) [[Grow larger culture quantity|1c a]] – the lab next door has a 15L fermenter you can borrow B) You decide to take [[anther approach|start]]

The commercial source is costly. Your PI approves the cost, but it will delay your progress by 4 weeks. Further, the commercial source is not purified to the extent you need. A) Wait for the commerical enzyme and proceed to [[purify it when it arrives|2]] B) Decide you can't risk waiting, because something else may also cause a delay and then you will miss your deadline and may lose your cool job as a Biochemist! [[Make another choice|start]] <img src="sad.png" width="200" height="200">

The synthetic DNA is not too expensive, but you will have to wait 3-5 weeks for it to arrive. You will then still need to subclone it, so you are really only skipping the PCR steps to make your own. Your PI counsels you to that this is not the best path, but it is still your choice A) [[Pick another option|start]] B) Go ahead, wait for the gene, and delay while you [[clone it into your vector|vector]] <img src="neutral.png" width="200" height="200">

There is still no response. Do you: A) [[Resend the request|1a a]] and continue to wait B) [[Choose again|start]] <img src="sad.png" width="200" height="200">

You have contacted someone else who might have the material. Unfortunately, they refer you to original investigator you are already waiting to hear from. Do you: A) [[continue to wait|1a b]] B) [[go back and choose another option|start]] <img src="sad.png" width="200" height="200">

Unfortunately, these primers are incorrect. [[Try again|1b]] <img src="sadder.png" width="200" height="200">

Congratulations! You correctly designed your primers and have performed the PCR reaction. You've taken the resulting product and analyzed it by agarose gel electrophoresis. <img src="happier.png" width="200" height="200"> Looking at the image of the gel, and knowing that you are expecting an enzyme of 310 amino acids, have you amplified your gene of interest? <img src="PCR.png" width="150"> A) yes! But there are multiple bands, you may want to optimize this reaction before [[you proceed|expression]] B) no! What the what?? [[Let me repeat this step|1b]] C) I think I'm going down a dark path, [[I want to make another choice|start]]

Congratulations, you've made it through the first hurdle! You should now have followed some path to arrive at having a sample of ParT enzyme that needs to be purified. [[Credits|Credits]] <img src="happier.png" width="200" height="200">

You are shocked to be reprimanded that you did not obtain the required Institutional Biosafety Committee approval for volumes of culture > 10 L. A formal warning has been issued that jeapordized the group's NIH funding if you proceed. You must now wait for approval until IBC meets. (Set: $mood = 2) A) That's OK, [[I'll wait|1c a2]] B) Never mind, I'll [[make another selection|start]] (If: $mood is 1)[<img width="200" height="200" src = "sadder.png">] (Elseif: $mood is 2)[<img width="200" height="200" src = "sad.png">] (Elseif: $mood is 3)[<img width="200" height="200" src = "neutral.png">] (Elseif: $mood is 4)[<img width="200" height="200" src = "happy.png">] (Elseif: $mood is 5)[<img width="200" height="200" src = "happier.png”>]

After waiting 4 weeks for the gene, it finally arrives. You subclone it into a vector, but it requires multiple tries. At this point you have invested a lot of time - you should put the "peddle to the metal" as it were to finish the project on the deadline. Proceed to try [[express|expression]] your recombinant ParT enzyme.

[[Still waiting|1c a3]]

[[Still waiting|2]]

You are expressing it from a recombinant vector using a T7 promoter system. You must first decide on the best way to run the protein expression: A) You will use a [[“cloning” strain of E. coli|wrongstrain]], like a DH5α strain. You grow this culture to an optical density at 600 nm of 0.7, induce protein expression with lactose, and hopefully express the protein for 6 hours at 37 °C. B) You will use a DE3 strain of E. coli, like a BL21 DE3 strain. You grow this culture to an optical density at 600 nm of 0.7, induce protein expression with [[lactose|lactose]], and hopefully express the protein for 6 hours at 37 °C. C) You will use a DE3 strain of E. coli, like a [[BL21 DE3 strain|ExpressionGood]]. You grow this culture to an optical density at 600 nm of 0.7, induce protein expression with IPTG, and hopefully express the protein for 6 hours at 37 °C. D) You decide to try an in-vitro translation system, where a commercially prepared extract of ribosomes and needed reagents are incubated with mRNA, and [[protein is produced directly|invitro]]. E) You decide to try expressing the protein in a [[yeast cell system|yeast]], in case there are important glycosylation events that will not be added in an E. coli expression system.

You have selected a strain of E. coli that does not have a T7 RNA polymerase, therefore no expression [[Go back and choose another option|expression]] <img src="sadder.png" width="200" height="200">

You have initial protein expression, but the yield is low because you chose to use lactose. The cells will metabolize this as a carbon source, so protein expression is short. You run the risk of not having enough protein in the end. A) [[grow more culture|1c a]] the same way as the first time to make sure you have enough B) continue on, running the risk [[you will run out of protein|2]] C) go back and [[choose another option|expression]] <img src="neutral.png" width="200" height="200">

You made a good choice. You may proceed to [[protein purification|2]]. <img src="happier.png" width="200" height="200">

This is an expensive option that produces low yields. Your PI is not happy and now you must go back and [[choose another option|expression]]. <img src="neutral.png" width="200" height="200">

This is a poor choice. The protein is originally from E. coli, therefore should not be glycosylated. [[Go back and make a better choice|expression]]. <img src="sadder.png" width="200" height="200">

Credits <a rel="license" href="http://creativecommons.org/licenses/by-nc-sa/4.0/"><img alt="Creative Commons License" style="border-width:0" src="https://i.creativecommons.org/l/by-nc-sa/4.0/88x31.png" /></a><br />This work is licensed under a <a rel="license" href="http://creativecommons.org/licenses/by-nc-sa/4.0/">Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License</a>. Conceived and created by Christina Bourne, with help from the <a href="http://xp.keeganslw.com/">eXperience Play workshop</a> Fall 2016 University of Oklahoma