【iGEMer分享】北京大学iGEM 2017年队员选拔考试题目

Bluepha作为一家由iGEMer创办的合成生物学企业，对于iGEM在中国年轻人中的传播和推广具有深厚的情怀。2016年Bluepha成立“ Bluepha iGEM支持平台 ”，全程支持了国内多支高校iGEM队伍。2017年，Bluepha想要陪伴更多iGEM队伍一起成长。我们希望把Bluepha微信和知乎公众号“蓝晶实验室”开放给所有iGEMer，将其作为国内iGEMer分享与发布的平台。

本期内容为“ iGEMer分享 ”系列的第一期。即将进入5月，国内不少高校iGEM队伍正面临着队伍建设和队员选拔的难题。在此，北京大学iGEM (Peking iGEM)委托“蓝晶实验室”独家发布《 北京大学iGEM 2017年队员选拔考试 》题目，希望能够与国内各个兄弟院校iGEM在队伍建设上进行深入的交流和讨论。

In this examination, you could get information, data and materials by any means if in need, except for discussing between examinees.

Answers in any form will be acceptable unless a specific rule is required in the question. Try your best to make your answers clear, solid and logical. To do this, additional figures, screenshots and photos of your handwriting are recommended.

Good luck to you all!

This part is for reviewing your involvement in brainstorming and the understanding of iGEM competition. Try to emphasize necessary details.

1. In the brainstorming, what part did you get involved in, and what’s your accomplishment? (6’)

2. Describe your group’s provisional project design. Divide it into several modules, and mark out some key milestones to be achieved, in English. (8’)

3. Describe your experimental design outlines for the milestones you write above. (6’)

4. How well do you know about other groups’ ideas? Make a description and comment for each one. If your current design was not chosen to be the final project of the team, how would you integrate/transform your current idea or design into the final project? (5’)

5. In your opinion, what is the judging standard of iGEM competition? What factors make an iGEM team a great team? (3’)

6. What kind of role do you expect yourself playing in the iGEM team? (2’)

7. ***Briefly describe your plan for June, July and August. How much time and energy can you put into the project? (Must answer)

This part is for testing your capacity of extracting information form previous researches and your knowledge on conducting molecular cloning experiments.

Read the attached paper Paired design of dCas9 as a systematic platform for the detection of featured nucleic acid sequences in pathogenic strains , which is a research achievement of Peking iGEM team 2015. Answer the following questions.

1. Briefly describe the design of the detection system. (2’)

2. Make a brief summary for each experiment set in this study (why, how, telling what). (6’)

3. Why is it that the design theoretically can improve the specificity as well as the sensitivity of nucleic acid detection? (6’)

Now, suppose you were the researcher.

4. What molecular cloning method(s) would you choose to build the chimeric proteins showed in Figure 2a, why? (4’)

5. Describe your molecular cloning process. Analyze its advantages and disadvantages. Try to make a comprehensive discussion. (6’)

6. Draw two sequence maps of chimeric protein Nfluc-dCas9 and Cfluc-FKBP. (6’) Requirements are as follows:

•The protein sequences should be carried on express vector pET28a.

•Necessary feature marking for different elements is required.

•Sequence maps should be in SnapGene file format (.dna).

•Primers must be contained in the map file to show your cloning methods.

•Not all elements for sequence construction are given. Find the rest by your own efforts (from supplementary material or iGEM parts registry).

Now, suppose you were working on improving this system.

7. As you can see in figure 3d, the detection system showed many non-ideal readouts when tested with purified E. coli genomic DNA. Why did the system generate a relatively high background readout with non-target DNA? (5’)

8. Based on existing results showed in the paper, what’s your idea for making further improvements on the sensitivity, as well as the specificity? (5’)

Notes: You need to do some basic literature research to answer question 7 and 8.

This part is for testing your capacity of modeling.

A

In single cell, concentration of gene product, as protein Y, is the result of the different between production rate (β) and degradation rate (α). The dynamic equation is given by

dY/dt =  β - αY

1. In the beginning, the protein production is at a constant rate β1. Suddenly, it shifts to β2. Calculate the protein concentration Y(t). (4’)

2. Function of gene is achieved by mRNA being translated, and in this process, mRNA itself can also be degraded by some regulator enzymes. Derive dynamical equation for the rate of change of mRNA and the change of protein. Assuming that mRNA is produced at rate of βm and degraded at rate of αm, and each mRNA molecule can produce ρ protein molecules in average over its lifetime. (3’)

3. Note that mRNA is degraded at a much faster rate than proteins (αm >> α).  Can this be used to form a quasi-steady-state assumption that mRNA levels are at steady state with respect to slower process? What is the effective protein production rate β in terms of βm, αm and ρ? (4’)

B

There are two species living in prairies, sheep and rabbit. Each species would grow to its carrying capacity in the absence of the other. This can be modeled by assuming logistic growth for each species. We assume that conflicts occur at a rate proportional to the size of each population. (If there were twice as many sheep, the odds of a rabbit encountering a sheep would be twice as great.) The conflicts reduce the growth rate for each species, but the effect is more severe for the rabbits.

The model which incorporates these assumption is as follow.

where

x(t) = population of rabbits

y(t) = population of sheep

What will happen in this dynamics? Draw a phase diagram. (8’)

C

The Michaelis-Menten kinetics (米氏动力学) has gradual (linear) dependence on the concentration, which means enzymes obeying Michaelis-Menten kinetics are not very good intracellular signaling switches. In 1981, Goldbeter A and Koshland DE Jr examined the transient and steady-state behavior of a reversible covalent modification system, and found that small percentage changes in the concentration of the effector controlling either of the modifying enzymes can give much larger percentage changes in the amount of modified protein.

This reaction is given by

While the input signal is defined as the concentration ratio of two enzymes ([E1]/[E2]), and the response is the concentration of W ([W] or [W*]), show this system will function as an ultra-sensitive switch. (9’)

D

Do simulation using MATLAB to show that the repressilator can display oscillations. (12’)

This part is for special skills like illustration and programming. Try to get bonus and show off your talent.

A

See attached iGEM_official_logo.png, convert this pixel graphic (像素图) into a vector graphic (矢量图) in .eps or .ai format, and change it into a red color theme.

B