Part:BBa_K2578610

The part BBa_K2578610 is the composite part of the Elsholtzia haichowensis metallothionein 1 synthesizing gene (EhMT1).
Elsholtzia haichowensis is a type of plants belonging to the family Labiatae. It is an indicator plant of copper mines and widely distributed in copper mining wastes and copper contaminated soils along the middle and lower streams of Yangtze River, China. [1]

Metallothioneins (MTs) are small cysteine-rich proteins made of 61-68 amino acids which can be found in a broad range of organisms, including both eukaryotes and prokaryotes. The MTs are expressed as intracellular protein. [2]

MTs are mainly responsible for metalloregulation in cells of living organisms. The rich Cys domains in MTs allow the non-covalent binding of trace metals such as cadmium, lead, copper and mercury, etc. [3,4]

The genomic sequence of the EhMT1 gene is obtained by reversing the mRNA sequence of the gene referenced from an academic paper by Yan Xia et. al.[5] into DNA sequence. The EhMT1 gene, registered as BBa_K2578611, is designed to be the composite part BBa_K2578610 by adding constitutive promoter J23100[6], Strong RBS B0032[7] and double terminator B0015[8].

The plasmid containing composite part BBa_K2578610 is ordered and synthesized by IDT. The ordered plasmid is transformed in E.coliTOP10 by heat shock. Transformed E.coli are used to perform a copper absorption test. The plasmid is also cloned into pSB1C3 backbone for part submission.

Assay : Test for copper absorption ability on engineered E.coli

Abstract of assay A

In preparation of this assay, transformed E.coli with MT plasmids (Homo sapiens and Elsholtzia haichowensis MT1 gene) and plasmid vector without insert (as control setup), are inoculated as E.coli colonies and incubated for another 18 hours in SOB medium. We then transferred the transformed E.coli in SOB medium to copper(II) sulphate solution, with concentration 2 mg/L and 10 mg/L, in a ratio of 1:9. E.coli was removed by centrifugation after 0 hours, 2 hours and 4 hours and 2.5 ml of supernatant was extracted to test for the absorbance after adding 175 ul of copper test solution. Using the standard curve produced by calibrating absorbance of solution against copper concentration in assay 3, we can estimate the copper concentration after incubation. Copper adsorption ability of the engineered E.coli can be studied from the information obtained.

Graph 1: Standard Curve of absorbance at 440 nm against copper concentration in SOB medium

We first conducted the assay with the 10mg/L copper(II) sulphate solution to determine the range of copper ion absorption by bacteria. E.coli transformed with Homo sapiens MT1 gene, Elsholtzia haichowensis MT1 gene are transferred to 10mg/L copper(II) sulphate solution after different incubation interval. And we used bacteria transformed with empty vector and SOB solution as control setups.

We measured the absorbance of the solution and the copper concentration is determined by using the standard curve.

Results

Table 1: Change of absorbance in 0, 2, 4 hours with engineered bacteria

Sample	Absorbance			copper concentration, mg/L
	0 h	2 h	4 h	0 h	2 h	4 h
Elsholtzia haichowensis MT1 gene	1.14	0.95	0.95	10.2	8.1	8.1
	1.11	1.02	0.96	9.8	8.9	8.2
Homo sapiens MT1 gene	1.14	1.02	1.01	10.2	8.9	8.8
	1.14	0.99	0.85	10.2	8.6	7.1
Empty Vector	1.14	0.98	0.94	10.2	8.5	8
	1.14	0.94	0.78	10.2	8	6.3
SOB medium	1.18	1.18	1.18	10.6	10.6	10.6
	1.18	1.18	1.18	10.6	10.6	10.6

Table 2: Copper absorption by engineered bacteria in 0, 2, 4 hours (10 mg/L)

Sample	Inital Concentration of copper, mg/L	Change of concentration, mg/L		% change of concentration
		after 2 hour	after 4 hour	after 2 hour	after 4 hour
Elsholtzia haichowensis MT1 gene	10.2	-2	-2	-19.90%	-19.90%
	9.8	-1	-1.6	-9.70%	-16.20%
Homo sapiens MT1 gene	10.2	-1.3	-1.4	-12.60%	-13.60%
	10.2	-1.6	-3.1	-15.70%	-30.40%
Empty Vector	10.2	-1.7	-2.1	-16.80%	-21.00%
	10.2	-2.1	-3.8	-21.00%	-37.70%
SOB medium	10.6	0	0	0.00%	0.00%
	10.6	0	0	0.00%	0.00%

Table 3: Average copper absorption by engineered bacteria in 0, 2, 4 hours

Sample	average initial concentration,mg/L	average chage of concentration, mg/L		average % change of concentration
		after 2 hour	after 4 hour	after 2 hour	after 4 hour
Elsholtzia haichowensis MT1 gene	10	-1.5	-1.8	-15%	-18%
Homo sapiens MT1 gene	10.2	-1.45	-2.25	-14%	-22%
Empty Vector	10.2	-1.9	-2.95	-19%	-29%
SOB medium	10.6	0	0	0%	0%

Graph 2: Effect of engineered bacteria on copper concentration

Graph 3: Average decrease in copper concentration after incubation with engineered bacteria

*Error Bar is standard deviation of estimated copper concentration

Graph 4: Percentage decrease in copper concentration after incubation with engineered bacteria

From the result, that is a significant decrease in copper concentration of the solution with E.coli with empty vector while comparing it to the SOB solution. While there is no change in the concentration for SOB medium, there is a decrease of 19% of copper concentration for E.coli after 2 hours of incubation and a decrease of 29% after 4 hours of incubation. Showing that E.coli itself are able to absorb copper ions.

Also, we could see that the copper concentration of the solution with Homo sapiens MT1 decrease 0.96 mg/L(14%) after 2 hours and 1.63 mg/L(22%) after 4 hours. Comparing with the control setup, we found the decreases of copper concentration in two setups are similar. Thus, we interpreted that the copper absorbed by E.coli transformed with Homo sapiens MT1 is similar to that absorbed by untransformed E.coli, meaning the present of Homo sapiens MT1 gene does not have significant effect in absorbing copper.

The copper concentration of the solution with Elsholtzia haichowensis EhMT1 decreased 1.49 mg/L(15%) within 2 hours and 3.02 mg/L(18%) within 4 hours. The concentration change of it and the control setup are similar so it is also interpreted that the copper absorbed by E.coli transformed with Elsholtzia haichowensis MT1 is similar to that absorbed by untransformed E.coli, meaning the present of Elsholtzia haichowensis MT1 gene does not have significant effect in absorbing copper.

To conclude, E.coli itself can absorb copper ions, E.coli with empty vector and transformed bacteria with the Elsholtzia haichowensis MT1 gene or Homo sapiens MT1 gene show similar copper ion absorbing ability.

Abstract of assay B

According to the World Health Organization’s guidelines for drinking water quality[1], 2 mg/L is the guideline value for copper concentration. We found the copper concentration would decrease around 2 mg/L after incubating the bacteria in 10 mg/L copper solution for 4 hours. We would like to know whether the concentration would decrease to 0 mg/L if the bacteria is incubated in 2 mg/L copper solution, or would the solution reach an equilibrium. Upon the question, we conducted the test of incubating transformed E.coli in 2 mg/L copper solution to investigate it.

Results

Table 4: Change of absorbance in 0, 2, 4 hour with engineered bacteria

Sample	Absorbance			copper concentration, mg/L
	0 h	2 h	4 h	0 h	2 h	4 h
Elsholtzia haichowensis MT1 gene	0.37	0.37	0.35	2.0	2.0	1.7
Homo sapiens MT1 gene	0.44	0.34	0.28	2.7	1.6	1.0
Empty Vector	0.37	0.34	0.34	2.0	1.6	1.6
Empty Vector	0.42	0.34	0.29	2.0	1.6	1.1

Table 5: Copper absorption by engineered bacteria in 0, 2, 4 hours(2 mg/L)

Sample	Initial concentration,mg/L	Change of concentration, mg/L		% change of concentration
		after 2 hour	after 4 hour	after 2 hour	after 4 hour
Elsholtzia haichowensis MT1 gene	2.0	0.0	-0.2	-0.0%	-10.9%
Homo sapiens MT1 gene	2.7	-1.1	-1.7	-39.4%	-63.0%
Empty Vector	2.0	-0.3	-0.3	-16.3%	-16.3%
Empty Vector	2.5	-0.9	-1.4	-34.2%	-55.6%

Graph 5: Effect of engineered bacteria on copper concentration (2 mg/L)

We conducted this assay with two cultures of transformed E.coli with empty vector. While there is a decrease of 0.3mg/L (16.3%) in the medium with an initial copper concentration of 2.0mg/L, there is also a decrease of 1.4mg/L(55.6%) in the one with an initial concentration of 2.5mg/L.

For the transformed bacteria with Elsholtzia haichowensis MT1 gene, there is a decrease of 0.2 mg/L (10.9%) after 4 hours of incubation in 2mg/L copper solution.

For transformed E.coli with Homo sapiens MT1 gene, there is a decrease of 1.1mg/L (39.4%) after 2 hours of incubation and 1.7mg/L(63.0%) after 4 hours of incubation in 2mg/L copper solution.

Table 6: Comparing copper absorption by engineered bacteria with different initial copper concentration

Sample	Initial concentration,mg/L	Change of concentration, mg/L		% change of concentration
		after 2 hour	after 4 hour	after 2 hour	after 4 hour
Elsholtzia haichowensis MT1 gene	9.8	-1.0	-1.6	-9.7%	-16.2%
	2.0	-0.0	-0.2	-0.0%	-10.9%
Homo sapiens MT1 gene	10.2	-1.6	-3.1	-15.7%	-30.4%
	2.7	-1.1	-1.7	-39.4%	-63.0%
Empty Vector(Set A)	10.2	-1.7	-2.1	-16.8%	-21.0%
	2.0	-0.3	-0.3	-16.3%	-16.3%
Empty Vector(set B)	10.2	-2.1	-3.8	-21.0%	-37.7%
	2.5	-0.9	-1.4	-34.2%	-55.6%

Graph 6: Decrease in copper concentration with different initial copper concentration for EhMT1 transformant

For transformed bacteria with Elsholtzia haichowensis MT1 gene. It shows similar percentage decrease of copper concentration when they are tested in different initial copper concentration. While there is a decrease of 1.60mg/L (16.2%) when the E.coli are incubated in 10mg/L copper solution for 4 hours, there is also a decrease of 0.23 mg/L (10.9%) for solution with E.coli incubated in 2mg/L copper solution for the same amount of time. It shows that the net absorption of transformed bacteria with Elsholtzia haichowensis MT1 gene stops when it reaches an equilibrium.

Graph 7: Decrease in copper concentration with different initial copper concentration for MT1A transformant

For transformed bacteria with Homo Sapiens MT1 gene. The difference of the percentage decrease of copper concentration when they are tested in different initial copper concentration is large. While there is a decrease of 3.09mg/L (30.4%) when the E.coli are incubated in 10mg/L copper solution for 4 hours, there is also a decrease of 1.70 mg/L (63%) for solution with E.coli incubated in 2mg/L copper solution for the same amount of time.

However, when we look at the data, the initial absorbance it show is 0.44 that the concentration of copper in the solution is 2.7mg/L according to the standard curve which it supposed to be 2.0mg/L. It shows that this set of data is unreliable. We hoped to conduct the assay again to obtain a more accurate set of data but we failed to do so due to time limit.

Graph 8: Decrease in copper concentration with different initial copper concentration for empty vector transformant

For the transformed bacteria with empty vector (Set A) it shows similar percentage decrease of copper concentration when they are tested in different initial copper concentration. While there is a decrease of 1.70mg/L (16.8%) when the E.coli are incubated in 10mg/L copper solution for 2 hours, there is also a decrease of 0.32 mg/L (16.3%) for solution with E.coli incubated in 2 mg/L copper solution for the same amount of time. It shows that the net absorption of transformed bacteria with empty vector (Set A) stops when it reaches an equilibrium.

For Set B of E.coli transformed with empty vector, the difference of the percentage change between the two different initial copper concentration is large, while there is a decrease of 3.83mg/L (33.7%) when the E.coli are incubated in 10mg/L copper solution for 4 hours, there is also a decrease of 1.38mg/L (55.6%) for solution with E.coli incubated in 2mg/L copper solution for the same amount of time. However, we notice that this set of data has a similar problem with the one of Homo Sapiens MT1 gene that the initial copper concentration deduced from the standard curve is much larger than it supposed to be. The concentration deduced from the standard is 2.5mg/L which the theoretical concentration is 2.0mg/L. Therefore, the data set B is unreliable, but we failed to perform it again due to time limit.

To conclude, the copper concentration does not decrease from 2 mg/L to 0 mg/L after 4 hours as we expected. Instead, we found that the copper concentration decreased by a similar percentage as the solutions did in the 10 mg/L test. Therefore, we can conclude that the net copper ion absorption stops when it reaches equilibrium.

Graph 9: Decrease in copper concentration with different inital copper concentration

Conclusion

Here are the few points that we would like to draw after the assays

1. Transforming plasmid with metallothioneins might not be able to enhance E.coli’s copper absorption ability.


2. By comparing the percentage of change in copper concentration between solutions with different transformed E.coli incubated, we cannot observe a significant difference between E.coli cloned with empty vectors, cloned with Elsholtzia haichowensis MT1 gene or Homo sapiens MT1 gene.


3. The reason behind might be the failure of expressing eukaryotic genes in prokaryotic cells such as E.coli.


4. By comparing the results from the assays using 2mg/L and 10mg/L, we found that the percentage change of copper concentration is similar between empty vector E.coli and transgenic E.coli, and both of them can absorb around 20-30% of copper ions after 4 hours of incubation.

Prospect

In the future, we would like to improve our device by lowering the copper concentration more effectively, causing a greater percentage decrease. Also, we aim to use the findings to design a device to absorb copper ions in water.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 529