Engineering Success | PIHS-Beijing - iGEM 2023

Engineering Success

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.

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 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

 

 

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.

 

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.

 

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.


 

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