iGEM 2023 — Team pihs-beijing

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {%
block lead %}Document the dates you worked on your project. This should be a
detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## Grand Meterial

---

**LB Culture(100mL):**
Trytone 1g
Extract 0.5g
NaCl 1g
(if solid) Agar 1.5g

## 8.3 Experimental Record

---

Topic：Plasmid Amplification
Researcher: Xinyi Zhao & Yuxuan Wu
Material：
plasmid——pQCXIP-mCherry (A+)
competent cell——Tran10
Methods/protocol：
Mix 100ng-500ng pQCXIP-mCherry with 100μl Tran10
Ice bath 30mins
Heat stimulation 42℃，90s
Ice bath 2-3 mins
        Spread-plate on A+ medium
Shaker, 37°C 200rpm，18h

[Design of primer: basic instructions]
Take pQXCIP & MO2 as an example
1.15bp overlap with MO2
2.include cleavage sites
3.18bp overlap

Wu: Example plasmid and primers

![Picture1](https://static.igem.wiki/teams/4971/wiki/picture1.png "Picture1")

5' ggccgcaccgccacctttggatccCACCatggtgagcaagggc 3' CG
per: 67% Tm=78°C
5' ggggagggagaggggcggaattcttacttgtacagctcgtc 3' CG per: 59% Tm=72°C

## 8.4. Discussion record

---

 **Topic: Discussion about following steps and
lactoferrin
    Reseacher:Xinyi Zhao & Yuxuan Wu**
1.Buy lactoferrin and verify that it has functions to retrain bacteria’s
growth
2.Search for method of bacteria counting
3.Two ways to free the bacteria from the effect of LTF: over-expression
of transferrin such as Feo family; add ferric or ferrous ions to the
medium
4.Use Coomassie blue stain or western blot to analyze

![Picture2](https://static.igem.wiki/teams/4971/wiki/picture2.png "Picture2")

![Picture3](https://static.igem.wiki/teams/4971/wiki/picture3.png "Picture3")

## 8.17 Experiment record

---

 **Topic: preliminary experiment
    Reseacher: Xinyi Zhao & Fangming Liu &
Zirui Fang & Zijun Yin & Yuxuan Wu**
Operation 1: Make LB medium, using autoclave to sterilization.
Operation 2: Do mock electrophoresis using retrovirus.
Operation 3：Calculate the concentrate, use ferric chloride to prepare
ferric ions solution.
Operation 4: Bacteria liquid inoculate.

## 8.18 Experiment record

---

 **Topic: preliminary experiment
    Reseacher: Zirui Fang & Yuxuan Wu**
Operation 1: adjust PH value of ferric solution by using sodium
hydroxide.
Result: Fail, sediment appeared.
Reason: sodium hydroxide was too concentrated. Carbonate or bicarbonate
solution should be used.

2.Prepare mole salt solution（MS）.Experiment aimed as the effect of LTF
effect and ferric ion impact on the effect.
Groups in experiment：/N，LTF，MS，LTF+MS，three repetition for each group
Result：The OD value for each group didn’t have much difference.However,
bacteria has obviously differnce in appearance. In the group of adding
LTF, dead bacteria clinged to the tubes’ wall. Mixing operation led to
the result of OD values. MS doesn’t have significant effect.

![Picture4](https://static.igem.wiki/teams/4971/wiki/picture4.png "Picture4")

Reason:The initial OD value and LTF concentration are
too high.Ferric ion concentration didn’t have variation. The initial OD
should be recorded.
Solution：use competence cells.divide one bacteria liquid into many，instead
of cultivating saperately. Ferric and ferrous ions concentration should
vary. LTF concentration should remain 1.2mg/3mL.Initial OD value should
range in 0.8-1.5.

## 8.21 Experimental record

---

 **Topic: cDNA preparation
Researcher: Fangming Liu**
Main：Use the high-expressed ltf mRNA （4uL/sample） of nasal mucosa cell,
offered by the lab,prepare the cDNA contains LTF sequece. Later design
primers for amplification of the cDNA.
Material：
1.mRNA sample x3
2.SuperRT retrotranscription kit
（1）SuperRT Reverse Transcriptase
（2）SuperRT Buffer
（3）dNTP mix
（4）Primer mix
（5）RNase free water
Steps：
1.Construct a reaction system（19uL/sample）
2.Put into Thermal Cycler （50min at 42C+ 10min at 85C，4C standby）
3.Using Ultramicrospectrophotometer to detect DNA concentration and
quality.
4.Preserve in -20°C environment.
Later steps：
1.Design primers
2.Design Sanger sequencing primer
3.PCR, amplifcation of each isoforms.
4.Design of full sequence Sanger sequencing primers
5.Construct plasmid
Sample 1（Rs0004R）：1014ng/uL
Sample 2（875811-pl）：994ng/uL
Sample 3（875811-GT）：923ng/uL
（260/280 and 230/260 conform to single strand DNA standard）

**See engineering and experiment part.**

## 9.16 experimental & discussion record

---

 Experimental design：
Use OD600=1.3 bacteria liquid to construct expression system. （2mL
bacteria liquid）
groups：
16°C：
1）IPTG 1:500
2）IPTG 1:1000
3）IPTG 1:2000
25°C：
4）IPTG 1:500
5）IPTG 1:1000
6）IPTG 1:2000
37°C：
7）IPTG 1:500
8）IPTG 1:1000
9）IPTG 1:2000
Fe2+ （T=25C，IPTG 1:1000）
10）20x dilution Fe2+
11）5x dilution Fe2+
12）no dilution
Fe3+ （T=25C，IPTG 1:1000）
13）20x dilution Fe3+
14）5x dilution Fe3+
15）no dilution
Without iptg（T=25C）：
16）standard concentration of Fe2+
17）standard concentration of Fe3+
18）controling group（E）：bacteria liquid（37C）

Specifically, based on the experiment of last time, the next step of
experiment is carried. 16°C，IPTG1:1000, ferric ion solution without
adding chelating agent or adjusting PH value, because the PH value of
the solution will not have huge impact on the whole system.
a）Fe2+（9.9mM）
19）100x dilution
20）50x dilution
21）20x dilution
22）10x dilution
23）5x dilution
24）standard
25）5x concentration
26）10x concentration
27）20x concentration
28）50x concentration
29）100x concentration
b）Fe3+（9.9mM）
same as Fe2+
（30-40）
 c）other（16°C） without IPTG
41）controling group2
42）Fe2+ 20x dilution only
43）Fe2+ 5x dilution only
44）Fe2+ 1x
45）Fe2+ 5x concentration
46）Fe2+ 20x concentration
Fe3+ （47-51、53）
The same as Fe2+
Add 10x dilution（50）
53) 20x dilution
52） 25°C controling group
In summary, there are 102 groups,additional with 10 MoE group.

（15-1）· 8= 112

material：
system：LB medium（Kanamycin+） 4mL/group
IPTG：
1:500 8uL/group
1:1000 4uL/group
1: 2000 2uL/group
Fe ions solution：1x group has final concentration of 0.1386mM
2mL system for 30mg LTF saturated concentration (27uL original solution)
(1x)
1x for 54uL original solution in 4mL system
Bovine LTF Standard

Result:
15:05 Inoculation in 250mL LB（K+）with 5mL ECS (1:50)
Kanamycin 1:1000
17:00 OD600=0.6
17:35 OD600=0.8
18:12 OD600=1
19:15 OD600=1.2

The western blot and Coomassie blue stain:

![Picture5](https://static.igem.wiki/teams/4971/wiki/picture5.png "Picture5")

![Picture6](https://static.igem.wiki/teams/4971/wiki/picture6.png "Picture6")

During the following experiments, we are disturbed with the problem that
controling groups show the expression of the LTF. There are
several explanation.
1.Operator leakage
a)Solution 1: change a bacterium species
b)Solution2: use CRISPR/Cas9 system to ensure the intact operator.
2.The cripsr system of the bacteria targeted the LTF sequence (because it is
detrimental) and cut it. (this can also explain why the protein is 30kD
shorter in the experiment of 9.10)

a)Solution 1: use CRISPR/Ca9 system to break all Cas protein in the
target backteria. (See part BBa\_K4971001 to BBa\_K4971007)

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{% extends "layout.html" %} {% block title %}Engineering Success{%
endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by
going through at least one iteration of the engineering design cycle.{%
endblock %}
{% block page\_content %}

#### Silver Medal Criterion #1

Demonstrate engineering success in a part of your project by going
through at least one iteration of the engineering design cycle. This
achievement should be distinct from your Contribution for Bronze.

If you plan to show engineering success by creating a new Part that
has been shown to work as expected, you must document your
contribution on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page)
for your team to be eligible for this criteria.

---

# Topic

In the
research part, we have multiple choices with brainstorm, such as
GFP nata de
coco, or producing antimicrobial peptide, or even degrade crude
oil to deal
with possible leakage. At last, we choose use E.coli to produce
human
lactotransferrin or lactoferrin(LTF), because of its high social
value and
possibility to produce. Details are in the description part and
human practice.

# Plasmid Design

We try to
design the plasmid. First, we find coding sequence of LTF from
NCBI. Second, we
find there are 4 isoforms of LTF. Because isoform1 and isoform2
are preprotein
and isoform3 lacks part of sequence, we choose isoform 4 as our
coding
sequence. Third, we ask the plasmid the lab has, Pet-28a, and
obtain it
sequence from Addgene. Fourth, we find the site to insert the
sequence and
insert them.

Next, we
modified the plasmid. We add the linkers to the plasmid(parts
BBa\_K4971011 and BBa\_K4971012).
Beside, his-tag is added. After that, we modified the CDS based
on the standard
provided by IGEM.

At last,
we send the sequence to Genescipt and synthesis the plasmid.

# Pre-experiment (constructing the expression system)

Transformed
Escherichia coli in kanamycin LB liquid medium was inoculated at
a
concentration of 1:50, and the bacteria culture was shaken at 37
° C and 200
rpm for 3 hours. The bacteria culture was sampled and its
optical density was
measured three times during the cultivation.

OD600
(1h30min) =0.545

OD600
(2h5min) =0.794

OD600
(2h50min) =1.3

The expression
system was constructed using OD600=1.3 concentration bacterial
solution (2mL
bacterial solution).

Grouping:

16 degrees
Celsius:

1) IPTG
1:500

2) IPTG
1:1000

3) IPTG 1:2000

25 degrees
Celsius:

4) IPTG
1:500

5) IPTG
1:1000

6) IPTG
1:2000

37 degrees
Celsius:

7) IPTG
1:500

8) IPTG
1:1000

9) IPTG
1:2000

Fe2+(T=25℃, IPTG 1:1000)

10) 20
times diluted Fe2+

11) 5 times
diluted Fe2+

12)
Concentration of Fe2+ original solution

Fe3+(T=25℃, IPTG 1:1000)

13) 20
times diluted Fe3+

14) 5x
diluted Fe3+

15) Fe3+
original concentration

No IPTG
group (T=25℃):

16)
Original concentration Fe2+

17)
Original concentration Fe3+

Control
group (E): Bacterial solution (37℃)

# Experiment and Result

The
successful experiment is based on the grouping shown above. The
bacteria
solution was cultivated under different temperature and IPTG
concentration conditions
and its optical density is listed below.

Blank
0.0

1. 16℃，IPTG 1：500; OD=0.989
2. 16℃，IPTG 1：1000;
   OD=1.020
3. 16℃，IPTG 1：2000;
   OD=1.040
4. 25℃，IPTG 1：500; OD=0.571
5. 25℃，IPTG 1：1000;
   OD=0.571
6. 25℃，IPTG 1：2000;
   OD=0.449
7. 37℃，IPTG 1：500; OD=0.418
8. 37℃，IPTG 1：1000;
   OD=0.465
9. 37℃，IPTG 1：2000;
   OD=0.462

------

1. 25℃，Fe2+ 0.05， IPTG 1：1000;
   OD=0.357
2. 25℃，Fe2+ 0.20， IPTG 1：1000;
   OD=0.473
3. 25℃，Fe2+ 1.00， IPTG 1：1000;
   OD=0.738
4. 25℃，Fe3+ 0.05， IPTG 1：1000;
   OD=0.492
5. 25℃，Fe3+ 0.20， IPTG 1：1000;
   OD=0.778
6. 25℃，Fe3+ 0.10， IPTG 1：1000;
   OD=0.508

------

1. 25℃，Fe2+ 1.00; OD=0.555
2. 25℃，Fe3+ 1.00; OD=0.441

------

1. 37℃; OD=1.811

![](https://static.igem.wiki/teams/4971/wiki/1-1.png)

Figure
1: photograph of the 37℃
experimental
group, cultivated at different IPTG concentration level.

As Figure 1
shows, the left most 37℃ control group is
significantly turbid.

![](https://static.igem.wiki/teams/4971/wiki/2-1.png)

Figure 2: photograph of the 16℃ experimental group,
cultivated at different IPTG concentration level.

It
could be observed that concentration levels remain the same
between the 16℃ group and
the previous-day initial concentration. Two speculations are
considered:

a.
the bacteria does not produce lactoferrin at 16℃.

b.
Balance was achieved between the rate of bacterial death and the
rate of
bacterial division.

![](https://static.igem.wiki/teams/4971/wiki/3.png)

Figure
3: photograph of the 25℃
experimental group, cultivated at different IPTG concentration
level.

Figure
3 demonstrates the results that the 25℃ group is close to elucidation,
indicating that the lactoferrin produced at this temperature has
a bactericidal
effect.

Firstly,
the control group data strikes within the expected range,
proving that the
experimental results are valid.

Secondly,
although no apparent trend could be extracted from the data in
the group with
iron addition, its survival rate is mostly higher than that of
the iron-free
control at 25℃.

Finally,
the groups without iron addition have an inhibitory effect on
bacterial growth,
which is directly proportional to temperature. However, the
inhibitory effect
of IPTG concentration is not significant.

According
to the original concentration of iron solution, the addition of
two non-expression
groups with iron addition has demonstrated antibacterial
effects, which can
prove that iron itself has an impact on bacterial activity
(oxidization effect
Fe3+>Fe2+). It can be speculated that
the effects of
iron itself, lactoferrin itself, and the combined effects of
iron and
lactoferrin all have their impacts on bacterial survival.

Specifically,
the bacteria under 16°C with
iron ions will produce more protein. The slowing rate of growth
in the group of
of adding IPTG only shows the product has the anti-bacteria
effect.

![](https://static.igem.wiki/teams/4971/wiki/4.png)
![](https://static.igem.wiki/teams/4971/wiki/5.png)

However, the product
is 30 kD smaller than expected, there are three possible
reasons:

1.
Glycoside modification
error

2.
E.coli stop the
expression because the product
is detrimental

3.
The plasmid is damaged
because of CRISPR

3.
After our analysis, we selectively altered certain conditions in our experiment and repeated our experiment 4-5 times

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{% extends "layout.html" %} {% block title %}Experiments{% endblock %}
{% block lead %}The materials and procedures we used{% endblock %} {% block
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{% extends "layout.html" %} {% block title %}Results{% endblock %} {%
block lead %}You can describe the results of your project and your future
plans here.{% endblock %} {% block page\_content %}

Results:

A comprehensive analysis of the results
shows
that, though we have achieved partial success in our goal, we have
some
questionable turnouts and need much more improvement.

Our current success comes from our
expression
experiment on September 9th (see notebook and Engineering success for
details).
The Western Blot results show that there are proteins with histidine
tags
present in groups where IPTG (expression inducer) and non in the
control
group(18). Paired with the decrease in bacterial OD values in the
corresponding
groups, we can confirm that we had produced functional lactoferrin
protein. The
groups with added iron ions displayed reduced inhibition towards
bacteria growth,
seeming to prove our hypothesis.

Problems arose when we attempted to
recreate the
experiment. Later experiments either produced strange results with
visible
bands in control groups during western blot or no bands at all. We
have come up
with several possible explanations for these abnormalities, noted in
our
notebook, and to be presented in our final video.

In pursuant to the laws of honesty and
sincerity
in scientific research, we cannot confidently claim that we have
achieved
success, only partial positive results among a lot of negative ones.
However,
this will not halt our steps in improving and trying until we see a
clearer
sky, as scientists should.

 Future
plans:

We have not yet given up on the dream
of
prokaryotic expression.

We plan do further our research into
previous
scientists’ work, and figure out what our problems are on a
theoretical level.
Then, we will take measures such as

1)
Changing bacterial strains
to
those more adept at facing lactoferrin (lactobacillus etc.)

2)
Adding tags onto our protein
to
solve solubility issues(seen as a possible way in papers)

3)
Inhibiting CRISPR-Cas
systems
to prevent damage or immunity in bacteria.

**Future
plans**

1.Experiment

Prior to October, members of our experimental group
conducted a series of experiments on human-derived lactoferrin isomers
(LTF) using methods such as controlled studies. Recently, we envisioned
broadening our project topics to ensure that our designs and products
can actually be effectively utilized in life. As a result, we have newly
designed some genes that can substantially solve many real-life problems
and will try to put them to the test in the coming January.

For example, the CRISPER system. Bacteria come with a
Crisper Cast System, which destroys viral DNA but may also cause damage
to our intelligence. Therefore, we designed a CRISPER system, which can
also be said to destroy the bacteria's original CRISPER system, to solve
this problem. The overall experiment is expected to be repeated twice in
October and a project to sequence the intelligence is underway. (See
Engineering for more details)

2.Lecture

For the knowledge popularization, we expect to launch a
total of 5 lectures online and offline to introduce the relevant
knowledge in detail, in order to achieve the effect of promoting the
popularization of science.

Activity time: October-November 23rd

Venue: Multi-functional classrooms in schools, Tencent
Meeting

Target Audience: School students

|  |  |  |
| --- | --- | --- |
| No. | Date/Time | Activity Subject |
| 1 | 10.16 16:00-16:30 | What exactly are lactoferrin isomers? |
| 2 | 10.19 17:00-17:30 | What are immunoglobulins? |
| 3 | 10.24 15:30-16:00 | What are whey proteins? |
| 4 | 10.30 16:00-16:30 | Cancer Awareness Campaign (i) |
| 5 | 10.31 16:00-16:30 | Cancer Awareness Campaign (ii) |

3.Online
Science Popularization

（1）Official Accounts

In order to expand our publicity for the popularization of
knowledge, in addition to the online and offline lectures, we expect to
organize and edit the contents of the lectures into public tweets and
send them out as original science content.

The content includes but is not limited to: introduction of
proteins such as lactoferrin, whey protein, and immunoglobulin,
knowledge of cancer and anti-cancer methods, and popularization of
biogenetic terminology related to our project topics.

（2）Videos

Interesting science videos are more intuitive than science
articles; people can learn more vividly in science videos. Therefore, we
plan to complete the production of two popular science videos: *What
is Lactoferrin* and *How Lactoferrin work.*

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