BIO Report

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Singapore​ ​Polytechnic
School​ ​of​ ​Chemical​ ​&​ ​Life​ ​Sciences
Diploma​ ​in​ ​Chemical​ ​Engineering
Biopharmaceutical​ ​Engineering​ ​(CP5074)
Experiment​ ​1​ ​report
Lecturer:​ ​Dr​ ​Jin​ ​Kai
Class:​ ​DCHE/ELE/FT/06
Group​ ​Members:
Full​ ​Name
Admission​ ​Number
Chai​ ​Zhi​ ​Xun
1614220
Andrew​ ​Lai​ ​Wen​ ​Qian
1613977
Goh​ ​Choon​ ​Shane
1614189
Guap​ ​Qing​ ​Yuen
1613696
WRITTEN​ ​ASSESSMENT
1.​ ​Answering​ ​BOTH​ ​questions:
(1) Explain the function of each type of agar medium you have formulated for
achieving​ ​your​ ​experimental​ ​objective.​ ​(10​ ​marks)
The first agar medium was formulated to ensure that the cells are able to grow in
normal cell culture medium. The second agar medium is formulated to ensure that
the plasmid DNA is present in the cells and ensure the segregational stability of the
cell. The third agar medium is formulated to ensure the cell’s structural stability. The
fourth agar medium is formulated to ensure that the cell has both structural and
segregational stability and thus can express the gene. The agar medium used are
(LB​ ​+​ ​A)​ ​and​ ​(LB​ ​+​ ​A​ ​+​ ​IPTG)
(2) Tabulate your colony counting results from various agar plates and
calculate the plasmid stability in terms of the percentage of cells containing
the​ ​correct​ ​foreign​ ​gene​ ​insert.​ ​(10​ ​marks)
Colonies
LB+A
LB+A+I
1
Does​ ​not​ ​glow
Does​ ​not​ ​glow
2
Glows
Glows
3
Glows
Does​ ​not​ ​glow
4
Glow
Does​ ​not​ ​glow
5
Does​ ​not​ ​glow
Glows
6
Glows
Does​ ​not​ ​glow
7
Glows
Glows
8
Does​ ​not​ ​glow
Glow
9
Glows
Does​ ​not​ ​glow
10
Glow
Does​ ​not​ ​glow
Cell that contains GFP will either glow in LB+A or LB+A+I. Since 9 out of 10
colonies has shown positive result, the percentage of cell containing the
foreign​ ​insert​ ​gene​ ​is​ ​90%.
2. Memo writing Write a memo (not more than 2 pages with Arial 12-point font
and 1-inch margins) to your superior, Mr Song, explaining your findings; and
provide appropriate recommendation(s) if any. You should attach suitable
pictures of your agar plates with developed colonies (not part of the 2-page
limit)​ ​to​ ​support​ ​your​ ​findings.​ ​(30​ ​marks)
To:​ ​Mr​ ​Song
Date:​ ​8/8/2017
Subject:​ ​Re-Selection​ ​of​ ​plasmid​ ​DNA
Dear​ ​Mr​ ​Song,
Our team is tasked to analyse the colonies produced by carrying out reselection.of
plasmid DNA. Therefore we selected 10 colonies for analysing. We used two agar
plates for selection. One contain ampicillin (LB+A), and the other with ampicillin and
IPTG​ ​(LB+A+I).​ ​The​ ​table​ ​below​ ​shows​ ​the​ ​result.
Colonies
LB+A
LB+A+I
1
Does​ ​not​ ​glow
Does​ ​not​ ​glow
2
Glows
Glows
3
Glows
Does​ ​not​ ​glow
4
Glow
Does​ ​not​ ​glow
5
Does​ ​not​ ​glow
Glows
6
Glows
Does​ ​not​ ​glow
7
Glows
Glows
8
Does​ ​not​ ​glow
Glow
9
Glows
Does​ ​not​ ​glow
10
Glow
Does​ ​not​ ​glow
Figure​ ​1.​ ​10​ ​colonies​ ​each​ ​on​ ​2​ ​agar​ ​plates,​ ​LB+A​ ​and​ ​LB+A+I
There​ ​are​ ​4​ ​different​ ​outcomes​ ​from​ ​the​ ​experiment​ ​as​ ​shown​ ​in​ ​figure​ ​1.
Firstly, the colonies that glow on LB+A but not LB+A+I, the lac suppressor and
pLysS plasmid is not working as intended, but the pET vector and pLysS are still
working correctly. This outcome is undesired as the colonies are unstable and we
are​ ​not​ ​able​ ​to​ ​control​ ​the​ ​protein​ ​induction​ ​time.
Secondly, the colonies that does not glow on both LB+A and LB+A+I means that the
cell contains the pLysS plasmid but it delete its GFP gene on the LB+A+I agar plate.
This​ ​is​ ​another​ ​outcome​ ​that​ ​is​ ​not​ ​desired.
Thirdly, the colonies that glows on both LB+A and LB+A+I means that the lac
repressor and the pLysS leaked and is not very effective. This is an undesired
outcome
Lastly, the colonies that does not glow on LB+A but glows on LB+A+I means that the
colonies contains the second plasmid and it also contain the GFP gene. It also
means that gene expression is carried out by the cell.This is the most desired
outcome.
In conclusion, our team recommend colony 5 and 8, which is the best colony to be
used​ ​for​ ​gene​ ​expression.
Regards,
Zhixun
Singapore​ ​Polytechnic
School​ ​of​ ​Chemical​ ​&​ ​Life​ ​Sciences
Diploma​ ​in​ ​Chemical​ ​Engineering
Biopharmaceutical​ ​Engineering​ ​(CP5074)
Experiment​ ​2​ ​report
Lecturer:​ ​Dr​ ​Jin​ ​Kai
Class:​ ​DCHE/ELE/FT/06
Group​ ​Members:
Full​ ​Name
Admission​ ​Number
Chai​ ​Zhi​ ​Xun
1614220
Andrew​ ​Lai​ ​Wen​ ​Qian
1613977
Goh​ ​Choon​ ​Shane
1614189
Guap​ ​Qing​ ​Yuen
1613696
WRITTEN​ ​ASSESSMENT
Writing​ ​Technical​ ​Report:
Draft a technical report (not more than 3 A4 pages with Arial 12-point font and
1-inch margins, excluding the cover page) for Mr. Song of GeneProducts Pte
Ltd, recommending with reasons, an optimized fermentation process for the
fermentation group to follow. You are required to use the format prescribed
below.​ ​(50​ ​marks)
Hypothesis​ ​and​ ​Objective
The​ ​objective​ ​was​ ​to​ ​formulate​ ​hypotheses​ ​regarding​ ​the​ ​effects​ ​of​ ​selected
fermentation​ ​parameters​ ​on​ ​cell​ ​growth​ ​and​ ​gene​ ​expression,​ ​construct​ ​appropriate
experimental​ ​designs​ ​for​ ​recombinant​ ​fermentation​ ​and​ ​conduct​ ​experimental
inquiries​ ​to​ ​either​ ​verify​ ​or​ ​nullify​ ​the​ ​hypothesis.​ ​Our​ ​hypothesis​ ​is​ ​that​ ​the​ ​smaller
the​ ​inoculum​ ​size,​ ​the​ ​greater​ ​the​ ​cell​ ​count​ ​density.
Material​ ​and​ ​Method
Material
● Shaker​ ​incubator
● Sterile​ ​shake​ ​flasks
● Sterile​ ​fermentation​ ​medium​ ​containing​ ​antibiotics
● Inoculation​ ​loop
● Microcentrifuge
● Spectrophotometer
● UV​ ​lights
● IPTG
Method
A.​ ​Setting​ ​up​ ​shaker​ ​incubators
1. ​ ​Change​ ​the​ ​existing​ ​set​ ​points​ ​for​ ​temperature​ ​and​ ​agitation​ ​speed​ ​if
necessary.
2. ​ ​Ensure​ ​the​ ​flask​ ​holders​ ​are​ ​firm.
3. ​ ​Operate​ ​the​ ​shake​ ​incubator​ ​to​ ​ensure​ ​the​ ​temperature​ ​and​ ​agitation​ ​speed
are​ ​controlled​ ​at​ ​set​ ​points.
B.​ ​Carry​ ​out​ ​inoculation
1. ​ ​Fill​ ​the​ ​sterile​ ​shake​ ​flasks​ ​(250​ ​ml)​ ​with​ ​50​ ​ml​ ​of​ ​sterile​ ​fermentation​ ​medium
containing​ ​antibiotics​ ​in​ ​the​ ​Biosafety​ ​cabinet.
2. ​ ​Aseptically​ ​transfer​ ​starter​ ​cultures​ ​to​ ​the​ ​shake​ ​flasks​ ​containing​ ​50​ ​ml​ ​of
fermentation​ ​medium​ ​(The​ ​inoculum​ ​size​ ​is​ ​designed​ ​by​ ​you).
3. ​ ​Fix​ ​each​ ​shake​ ​flask​ ​with​ ​the​ ​flask​ ​holder​ ​inside​ ​the​ ​shake​ ​incubator.
4. ​ ​Close​ ​the​ ​door​ ​and​ ​start​ ​the​ ​cell​ ​cultivation.
C.​ ​Perform​ ​cell​ ​induction
1. ​ ​Take​ ​samples​ ​from​ ​shake​ ​flask​ ​cultures​ ​for​ ​OD​ ​(600nm)​ ​measurement.
2. ​ ​Cells​ ​are​ ​induced​ ​with​ ​IPTG​ ​at​ ​the​ ​determined​ ​final​ ​concentration​ ​(designed
by​ ​you)​ ​when​ ​the​ ​OD​ ​(600​ ​nm)​ ​reaches​ ​a​ ​specific​ ​value​ ​(designed​ ​by​ ​you).
3. The​ ​temperature​ ​set​ ​point​ ​of​ ​the​ ​shake​ ​incubator​ ​may​ ​be​ ​changed​ ​to​ ​a​ ​new
value​ ​(designed​ ​by​ ​you).
4. The​ ​flasks​ ​are​ ​placed​ ​back​ ​to​ ​the​ ​shaker​ ​incubator​ ​and​ ​grow​ ​for​ ​a​ ​period
determined​ ​in​ ​your​ ​own​ ​protocol.
D.​ ​Analyze​ ​cell​ ​growth​ ​and​ ​gene​ ​expression
1. ​ ​1​ ​ml​ ​of​ ​cell​ ​culture​ ​is​ ​taken​ ​for​ ​OD​ ​(600​ ​nm)​ ​measurement​ ​using​ ​a
spectrophotometer.
2. 1​ ​ml​ ​of​ ​cell​ ​culture​ ​is​ ​centrifuged​ ​at​ ​14,000x​ ​g​ ​in​ ​a​ ​microcentrifuge​ ​for​ ​2
minutes,​ ​and​ ​supernatant​ ​is​ ​discarded.
3. The​ ​cell​ ​pellet​ ​is​ ​observed​ ​under​ ​UV​ ​lights​ ​and​ ​saved​ ​in​ ​a​ ​freezer​ ​(20oC)​ ​for
subsequent​ ​SDS-PAGE​ ​analysis.
Results​ ​and​ ​Discussion
Inoculum​ ​Size
Time
Cell​ ​Count​ ​Density
Low
1000
0.02
1100
0.36
1200
0.54
1000
0.20
1030
0.56
1130
0.7214
High
Conclusion
The​ ​greater​ ​the​ ​inoculum​ ​size,​ ​the​ ​higher​ ​the​ ​cell​ ​density.​ ​As​ ​seen​ ​from​ ​the​ ​results,
cell​ ​density​ ​of​ ​high​ ​inoculum​ ​size​ ​is​ ​larger​ ​compared​ ​to​ ​when​ ​inoculum​ ​size​ ​is​ ​low.​ ​A
possible​ ​reason​ ​for​ ​this​ ​could​ ​be​ ​related​ ​to​ ​the​ ​time​ ​that​ ​the​ ​cell​ ​medium​ ​stays​ ​in​ ​the
reactor.​ ​With​ ​lower​ ​inoculum​ ​size,​ ​the​ ​cell​ ​count​ ​density​ ​after​ ​fermentation​ ​should​ ​be
higher​ ​than​ ​that​ ​of​ ​high​ ​inoculum​ ​size​ ​as​ ​lesser​ ​toxins​ ​are​ ​produced​ ​and
accumulated.​ ​However,​ ​in​ ​our​ ​case,​ ​the​ ​cell​ ​density​ ​is​ ​lower.​ ​This​ ​may​ ​be​ ​due​ ​to​ ​the
fact​ ​that​ ​the​ ​fermentation​ ​process​ ​in​ ​both​ ​cases​ ​did​ ​not​ ​finish​ ​and​ ​is​ ​stopped.​ ​Thus,
the​ ​fermentation​ ​in​ ​both​ ​cases​ ​did​ ​not​ ​reach​ ​the​ ​stationary​ ​phase​ ​and​ ​is​ ​stopped​ ​at
the​ ​log​ ​phase.
Recommendations​ ​for​ ​Future​ ​Work
We​ ​recommend​ ​that​ ​for​ ​future​ ​inoculation,​ ​prepare​ ​a​ ​few​ ​samples​ ​to​ ​be​ ​inoculated
with​ ​loops​ ​of​ ​different​ ​sizes​ ​to​ ​find​ ​the​ ​relationship​ ​between​ ​the​ ​various​ ​inoculation
loop​ ​size​ ​and​ ​the​ ​cell​ ​count​ ​density​ ​to​ ​find​ ​the​ ​optimal​ ​loop​ ​size.
Singapore​ ​Polytechnic
School​ ​of​ ​Chemical​ ​&​ ​Life​ ​Sciences
Diploma​ ​in​ ​Chemical​ ​Engineering
Biopharmaceutical​ ​Engineering​ ​(CP5074)
Experiment​ ​3​ ​report
Lecturer:​ ​Dr​ ​Jin​ ​Kai
Class:​ ​DCHE/ELE/FT/06
Group​ ​Members:
Full​ ​Name
Admission​ ​Number
Chai​ ​Zhi​ ​Xun
1614220
Andrew​ ​Lai​ ​Wen​ ​Qian
1613977
Goh​ ​Choon​ ​Shane
1614189
Guap​ ​Qing​ ​Yuen
1613696
Answer​ ​ALL​ ​questions:
1. State the chemical compositions for each solution used in the protein
purification​ ​protocol​ ​designed​ ​by​ ​you.​ ​(15​ ​marks)
1.​ ​Sodium​ ​phosphate​ ​stock​ ​solution,​ ​5x​ ​(100ml)
-500mM​ ​NaH​2​PO​4​,​ ​50mM​ ​Tris​ ​Cl,​ ​pH​ ​8.0
2.​ ​1M​ ​imidazole​ ​solution(50ml)pH​ ​7.0
3.​ ​Deionised​ ​water
Wash​ ​buffer
1.​ ​5ml​ ​of​ ​sodium​ ​phosphate​ ​stock​ ​solution
2.​ ​45ml​ ​of​ ​deionised​ ​water
Elution​ ​buffer
1.​ ​10ml​ ​of​ ​wash​ ​imidazole​ ​solution
2.​ ​5ml​ ​of​ ​sodium​ ​phosphate​ ​stock​ ​solution
3.​ ​45ml​ ​of​ ​deionised​ ​water
2. Resolve the following issue: Supposed that one of your team members, Mr.
Siew Huat Tat, approached you one day and said, “Supposed, hypothetically –
I mean just for discussion purposes – that a staff from our company is
approached by a head-hunter to work for a competitor. Do you think this staff
can share some of his knowledge about this company with the competitor?
After all, the offer by the competitor is very attractive, let’s say several
thousand dollars of one-time joining fee, and up to $800 more in monthly
salary than what one can get in GeneProducts. In any case, I think in this
business, a competitor can always come up with similar products or protocols
very quickly, so – again, hypothetically – the risk to this company from anyone
sharing insider knowledge or secret is quite minimal. What is your view on
this?” You suspected that Mr. Siew Huat Tat is in fact referring to himself, and
that the competitor is none other than Good Genes Pte Ltd, who is known to
have invested in novel protein purification techniques. In fact, Mr. Song is
quite weary of the company, as it had successfully lured one of GeneProducts’
research scientists to join the company 2 months earlier. Good Genes had
been aggressively expanding its presence in the region, and more poaching of
staff can be expected. In addition, you and your team had not signed any
document pledging secrecy on the new protein purification protocol that your
team developed; so technically, before anything was signed, one is not
prohibited by company regulations to divulge any important information that
you might possessed. Explain your views to Mr. Siew Huat Tat (not more than
1 A4 page with Arial 12- point font and 1-inch margins) with relevant code of
ethics​ ​(such​ ​as​ ​that​ ​in​ ​IChemE,​ ​or​ ​AIChE)​ ​to​ ​support​ ​your​ ​answer.​ ​(15​ ​marks)
Personally, I do not think that the stuff should share his knowledge about the
company to the competitor company. Even though the protein purification protocol
developed has not been signed pledging secrecy, it is the company’s property and
the company has the right to sue anyone who shares this protocol without consent.
Furthermore, in the competitive market of biopharmaceuticals, the company has to
have an edge over other companies. Even though it might be true to say that other
companies can come up with protocols very quickly, a slight edge over other
company might translate to increased revenue gained by the company. Lastly,
sharing of the protocol might cause you to lose your credit as the competitors can
easily​ ​claim​ ​this​ ​technology​ ​or​ ​protocol​ ​as​ ​theirs.
PowerPoint Slide Preparation Mr. Song asks you to prepare a set of
PowerPoint slides to brief senior management about your newly developed
purification protocol. Mr. Song added: “These are all very busy people, and
they may not fully understand the concepts. Try keeping your slides to a
minimum, using simple terminology. I suggest that you do not use more than
10 slides, excluding the first opening slide and the last closing “Thank You”
slide.” Note: You should name your file as CP5074_E5_XX_Y.PPT; where XX is
your class and Y is your group number, and submit through email. You
submission will be graded for both technical accuracy and good slide design.
(20​ ​marks)
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