Part:BBa_K3156000

pLac Promoter - sfGFP

This part consists of a inducible pLac promoter and a sfGFP coding sequence which can be used for quantitative measurement of base-editing efficiency.

Design

This part is improved from iGEM part BBa_K864400

Characterization

In our design, the induction signal will be detected and stored in the plasmid DNA sequence of our genetically modified E. coli. The bacteria will be gathered from the capsule after it left human body and sent to lab for further quantitative analysis in order to represent the inflammation level in colon. Therefore, in our project, the fluorescence intensity will be the index that represents levels of gut inflammation.
Since then, we first needed to develop a standard quantitative measurement protocol, so we studied the following articles:[1] Zong, Y et al (2017)[2] and Zhang, H. M. et al(2015).

Figure 1.We constructed our reporter circuit with sfgfp and pTac promoter

Following reference, we determined the experimental procedure:
Flat-bottom 96-well plates and sealing film were used throughout the study. Bacteria harboring parts/circuits of interest were inoculated from plates to LB medium and grown overnight (8−12 h, 1000 rpm, 37 °C, mB100-40 Thermo Shaker). Ten microliters of each overnight culture was sequentially diluted into 130 μL of fresh medium twice; the total dilution fold was 196. After growing the diluted cultures for ∼3 h, we diluted the exponentially growing cultures 700-fold using fresh medium; the dilution process was as follows: 10 μL of cell culture is added to 130 μL of M9 medium, which is followed by diluting 3 μL of this into 147 μL. Then, cultivation continued (1000 rpm, 37 °C, mB100-40); atspecifictimepoints,a2−50μL aliquot of each culture was transferred to a new plate containing 200 μL of PBS with 2 mg/mL kanamycin preadded to terminate protein expression. For the time course of cell growth after 700-fold dilution, OD 600 was recorded using Varioskan Flash (Thermal Scientific); the time interval was 5 min.

Figure 2.Experimental procedure for recording the time course of gene expression using flow cytometry.

The graph below shows our result.

Figure 1.Ptac response curve in medium copy number vector.

Characterization by 2021 iGEM BNDS_China

In our 2021 project, we intended to optimize the rhlABC genes for the synthesis of rhamnolipids. To keep extraneous variables constant and help us to quantitatively analyze the yield of rhamnolipid, we adopted the part BBa_K3156000, in which the rate of expression can be controlled by adding IPTG to relief the suppression on the pTac promoter. But beforehand, we need to first quantitatively characterize the part to ensure its functions.
The original part consists of a gene fused with lacI and pTac, we used sfGFP to operationalize the strength of expression when induced with different concentration of IPTG since sfGFP can be easily quantified through flow cytometry or with a plate reader yet the amount of rhlABC enzymes can only determined indirectly by measuring the concentration of rhamnolipid with HPLC-MS.

Method

After the plasmid is transformed into Escherichia coli (DH5α), a single colony was selected and inoculated in 5 ml ampicillin-added-LB at 37° C, 220 rpm for 8 hours. Then 10 microliter of the seed culture was added into 1 mL LB in each well on a plate, diluting it at 100 folds. After 8 hours of inoculation at 37° C, 380 rpm, 1 microliter of the culture was added into 300 microliters of LB induced by IPTG.
This final culture was shook at 380 rpm at 37° C for 8 hours. After inoculation, it was measured for its OD and fluorescence using a plate reader. The excitation wavelength is 488nm, the emission wavelength is 512nm, and the gain is manually set at 40. In the following graph are the results.

Figure 3.concentration of IPTG added. In the control group, no bacteria culture was added .

Analysis

From the graph we can see that the fluorescence of the culture ranged from less than 600 a.u. to about 780 a.u. This supports the previous characterization data where the derivative increases between 10 μM and 500 μM. In this experiment, we expanded the range of IPTG concentration up to 10 mM, but the curve gradually flattens out and no longer showed significant increase from 1 mM IPTG to 10 mM IPTG. Therefore, we can infer that the suppression on pTac promoter is totally relieved when about 1 mM of IPTG was added. More IPTG cannot increase its expression rate and the toxicity of IPTG could possibly influence the microbes.


A flaw is that the error is relatively large, probably due to the method used to prepare the sample. As the sample was collected, there’s visible sediments in the bottom of the wells, which can affect the accuracy of the results. To solve this problem, a higher rpm and shorter culturing time can be considered. Even though the accuracy is not ideal, we applied the ANOVA test and attained p-value smaller than 5% between point 1, 2, 3, 4 and 5. The p-value is greater than 5% between point 6, 7, 8, point 9, 10, 11. This further supports our conclusion: when IPTG exceeds 1mM, there’s no significance increase in the expression rates.

Figure 4.Fluorescence of sfGFP graphed against IPTG concentration on a logarithmic scale. A non-linear regression (logarithmic growth) curve was fit into the graph.

Reference

[1]Zong, Y., Zhang, H. M., Lyu, C., Ji, X., Hou, J., Guo, X., ... & Lou, C. (2017). Insulated transcriptional elements enable precise design of genetic circuits. Nature communications, 8(1), 52.
[2]Zhang, H. M., Chen, S., Shi, H., Ji, W., Zong, Y., Ouyang, Q., & Lou, C. (2015). Measurements of gene expression at steady state improve the predictability of part assembly. ACS synthetic biology, 5(3), 269-273.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 170