During the communication with team “Purdue”, we knew that they prepare to do an in vitro colonization assay to see if their genetically modified E. coli could survive in the target place. Inspired by their thought, we also designed a series of in vitro experiments in order to investigate if our constructed bacteria could survive in gastrointestinal tract.
Two parts of experiments were
designed. One of them is to
investigate if our genetically modified E. coli could adapt the physical environment of
gastrointestinal tract. We cultured the bacteria in artificial gastral juice and
intestinal juice, plotted the growth curve and compared with strains cultured in LB.
Another part is to test if our bacteria had the ability to compete with the native
microbiota in gastrointestinal tract. Based on such an idea, we obtain the feces of mice
from College of Pharmacy in our university and coculture them under different
conditions. Competent cells of MC4100 were made based on TSS method and transformed by
KCM transformation method (see notebook in detail).
Main materials used in these experiments are shown in Table 1. Related equipment is similar to InterLab study and is not shown here, with acidimeter (LEICI PHSJ-3F) and balance being the additional ones.
|Competent cells||Trans 5 alpha form|
|DNA||2017 kit plate 7|
|Yeast extract||OXOID LP0021|
|Agar||Solarbio biotechnology grade|
|Trypsin 1:300 from porcine pancreas||BBI Life Science|
|Pepsin 1:3000 from porcine stomach||BBI Life Science|
Firstly, we prepared
competent cells of MC4100 based on TSS
method and transformed competent cells with plasmid bearing BBa_J04450 by KCM
Artificial gastral juice and intestinal juice were prepared according to the literature (Table 2). Due to our constructions were in different strains, we tested the growth condition of them respectively. mRFP (BBa_J04450) was used as reporter.
We first transformed the competent DH5 alpha and MC4100 with plasmid provided in Competent Cell Kit, cultivated them under 37 degrees centigrade overnight. Then single colonies were picked based on colony color. Liquid culture was made and incubated overnight. After incubation for about 12~16h, culture was sampled and original OD600 was measured. Then culture was diluted until OD600 = 0.02, which relied on calculation excel of InterLab measurement offered on official website. 1mL Diluted culture was allocated into a 10mL tube mixed with 1mL artificial gastral juice or intestinal juice. Nine identical tubes were prepared to do the test at an interval of 3 hours from time 0 to time 24 hours. Three biological replicates were made and growth curves were plotted after measurement. A control group cultivated in 2mL LB was manipulated in the same way. Corresponding concentration of Chloromycetin was premixed in the culture medium.
According to our results, strain MC4100 grows
relatively better than DH5 alpha in pure LB. Compared with culture in pure LB, it is
easy to see that the growth of constructed strain will be influenced in either
artificial gastral juice or artificial intestinal juice. We can also get the conclusion
that MC4100 is more sensitive to artificial gastral juicy rather than intestinal juice,
probably suggesting its vulnerability to acidic environment. In general, our data shows
that the constructed bacteria can survive in an environment mimicking gastrointestinal
tract to some extent, though their growth will be affected.
Feces of mice were obtained from College of Pharmacy with the help of Qian Jiang (see attribution). 0.25g feces were dissolved in 2mL LB medium with no antibiotic and cultured overnight. Strains mentioned before were cultured with corresponding antibiotics. After incubating for about 14h, Feces cultures were then filtered by normal filter paper (not filters with diameter equaling to 0.2 micron) to get supernatant. OD600 of Supernatant as well as constructed strain cultures were measured later. Based on measurement result, cultures were diluted to OD600 = 0.02 according to calculation excel provided on official website. Supernatant and bacteria culture were than mixed in various ratio. Mixtures were cultured in either artificial gastral juice or intestinal juice overnight. Then we did gradient dilution to the culture and spread them on plates with no antibiotic. Plates were incubated under 37 degrees centigrade and observed after 2 days.
We sadly found that except the positive control (ratio 1:0 in figure 4, 5), none of experimental plate became even reddish, not to say become as red as positive control after incubation for 2 days. Maybe exposure to atmosphere dramatically accelerated the growth of some bacteria which grew worse in gastrointestinal tract with few air. Probably coculture in an anaerobic environment may provide different result.