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BBa_K3796000 BBa_K3796203 BBa_K3796204

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Engineering Success

CAU_China has spent great efforts in biological engineering this year. Here we present three of our new parts that has been proved to work as desired through our engineering process, respectively BBa_K3796000, BBa_K3796203 and BBa_K3796204. You can also find more about our engineering in Results .

BBa_K3796000: Corynebacterium glutamicum strong constitutive promoter P0864

1st iteration

• Aim: This is a constitutive promoter in Corynebacterium glutamicum which had little information previously on the Registry. We use this promoter to control the expression of the γ-PGA synthetase genes in our project. Therefore, we need to test its strength in C. glutamicum first. Also, to fully characterized this new part, we also want to know if it works as well in the typical chassis E. coli.


• Design: We want to solve this by inserting a reporter gene behind P0864 to visually show its strength as a promoter. For convenience, we choose the fluorescent protein mCherry, whose excitation light and emission light both fall into the range of visible light. Also, we choose to use the shuttle vector pXMJ19 that is applicable to both E. coli and C. glutamicum. The genetic circuit we used as a device in testing is BBa_K3796219 and the blank control is BBa_K3796220. Particularly, you may notice that there is a tac promoter upstream of the MCS on pXMJ19, so a proper blank control to exclude the influence of Ptac leaky expression is needed. Ideally, the fluorescence intensity/OD₆₀₀ should be higher in experiment group than in the control group.
  
  
  

  Fig.1 Genetic circuit for testing the strength of P0864

  
  
  
  

  Fig.2 Genetic circuit for blank control



• Build: We built the genetic circuits using Clonexpress^® Multi One Step Cloning kit from Vazyme to connect RBS sequence (BBa_K3796001, synthetic) and mCherry gene (BBa_J06504) downstream to P0864. We successfully constructed the above sequences and verified them by colony PCR and sequencing.
  
  
  

  Fig. 3 Agarose gel map of BBa_K3796219 circuit.
  a.Lane 1 to lane 9 are electrophoresis bands of BBa_K3796219 circuit, where lane 1 to lane 4 are for transformed E. coli, lane 5 to lane 9 are for transformed C. glutamicum. b. Lane 1 (E. coli) and lane 2 (C. glutamicum) are electrophoresis bands of BBa_K3796220 circuit



• Test: In the 1st iteration, we tested the strength of P0864 by simple qualitative test of the fluorescence under the excitation light of mCherry protein (580nm).
  
  
  

  Fig. 4 The difference of fluorescence in Escherichia coli (including experimental group (left) and control group (right)).
  a. Image under excitation light. b. Image under natural light.

  
  
  
  

  Fig. 5 Difference of fluorescent proteins expressed by Escherichia coli and Corynebacterium glutamicum.
  a. Images of Corynebacterium glutamicum after centrifugation, including experimental group (left) and control group (right). b. Images of E. coli after centrifugation, including experimental group (left) and control group (right).

  As you can see, compared with control group, the experiment group apparently showed red fluorescence, both under the natural light and excitation light. However, qualitative tests are not enough for the characterization of the strength of P0864, and we further wanted to know in which chassis did it work better.



• Learn: We learned that P0864 could work in E. coli and C. glutamicum. The comparison of its strength between these two chassis needed to be further tested by other methods.

2nd iteration

• Re-Test: We designed a quantitative test to better characterize this promoter in different chassis. After the bacteria had been cultured at 37 ℃ for 26 hours, the fluorescence was measured with a fluorescence spectrophotometer and OD₆₀₀ was measured with a visible spectrophotometer. We use the ratio of fluorescence intensity/OD₆₀₀ to reflect the relative fluorescence intensity under the unit absorbance value.
  
  
  

  Fig.6 After E. coli and C. glutamicum were cultured at 37 ℃ for 26 hours, the ratio of measured fluorescence intensity data to OD₆₀₀ data.
  (the excitation light of 580nm and emission light of 610nm)

  T-tests were performed to show that there were significant difference between experiment group and control group. Plus, this difference is bigger in C. glutamicum.We have done four repeats and the standard deviation is acceptable.



• Re-Learn: P0864 is a constitutive promoter that works both in E. coli and C. glutamicum. Its strength tends to be stronger in C. glutamicum. If used in E. coli , codon-optimization may be carried out.

BBa_K3796203: ndoA (endoribonuclease toxin in Bacillus subtilis)

1st iteration

• Aim: We aim to test if the over-expression of the toxin gene ndoA, originally from Bacillus subtilis can kill Corynebacterium glutamicum effectively as well.
• Design: The genetic circuit we used as a device in testing is BBa_K3796217. We continued using the shuttle vector pXMJ19, since it has an inducible promoter, tac promoter. We inserted the gene ndoA between the tac promoter and rrnB terminator on the plasmid, in order to test the effect of ndoA with the induction of IPTG. Ideally, after adding IPTG, the growth of bacteria should be greatly depressed compared to the group without induction.
  
  
  

  Fig. 7 Genetic circuit for ndoA verification

• Build: We used ClonExpress II one-step cloning kit (Vazyme Biotech, China) to build the circuit for testing. The expression vector was transformed into E. coli DH5α first and then into C. glutamicum by electroporation. Electrophoresis showed that we had built the circuit successfully.
  
  
  

  Fig. 8 The agarose gel electrophoresis of enzyme-digested product. (a)(Hind Ⅲ) and colony PCR (b)(E. coli)..
  Lane(a): MK:Marker; 1:pXMJ19-ndoA digested by Hind Ⅲ; Lane(b): MK:Marker; 1:Product of colony PCR (pXMJ19-ndoA in E. coli)

  
  
  
  

  Fig. 9 The agarose gel electrophoresis of colony PCR (C. glutamicum).
  Lane: MK:Marker; 1,2:product of colony PCR (pXMJ19-ndoA in C. glutamicum)(Targets are respectively endogenous mazF and ndoA); 3,4:negative control

• Test: To design a quick qualitative test, ‘divided plate’ assay was carried out at the beginning. We divided a plate with LB medium into four parts and used the spread plate method to see if ndoA can kill C. glutamicum and this killing effect is not caused by the toxicity of IPTG. Bacteria carrying empty vector was added into 1 Ep tube and bacteria carrying the gene circuit was added into 2 Ep tubes with 1.8 mL LB liquid containing 10 μg/mL chloramphenicol respectively. After incubating the culture in a shaker at 30 °C, 220 rpm until OD₆₀₀ reached 0.6, we prepared LB plates that were divided into four quarters marked A,B,C,D. We spread the diluted bacteria solution(10^-3) on the quarters respectively, and all plates were incubated at 30 °C for a certain time.
  
  
  

  Fig. 10 Divided Plate Assay
  Quarter A: C. glutamicum carrying empty vector and 0.8mM IPTG was added; Quarter B: C. glutamicum carrying empty vector without adding 0.8mM IPTG; Quarter C: C. glutamicum carrying the vector pXMJ19-ndoA without adding 0.8mM IPTG; Quarter D: C. glutamicum carrying the vector pXMJ19-ndoA vector and 0.8mM IPTG was added

  Comparing Quarter A and Quarter B, we can see that the growth condition of the two is very similar, which means that the toxicity of 0.8mM IPTG is very low and it hardly kills C. glutamicum. Comparing Quarter C and Quarter D, we can see that there is apparently less bacteria survived in Quarter C, and the diameter of the colonies in Quarter C is rather small as well. As for the comparison between Quarter B and Quarter D, we assume that there is serious leaky expression of ndoA controlled by tac promoter in Quarter D, which deteriorates its growth condition. We repeated this experiments for 5 times and got nearly the same result.

• Learn: We got the preliminary conclusion that ndoA does have a killing effect on C. glutamicum. However, without controlling the induction time, it’s hard to get a satisfying result because the leaky expression of the tac promoter.

2nd iteration

• Re-Test: To give a further quantitative test and took leaky expression into consideration, we carried out CFU assay to characterize the killing effect of ndoA instead of determining OD₆₀₀ in order to get rid of dead bacterial cells. CFU was quantified by counting the colonies on one plate and normalizing the number to volume of 1 mL culture. We added 0.8mM IPTG as OD₆₀₀ reached 0.6, and estimated CFU by spreading 25μL of the culture on 3 LB solid medium every hour after the induction with 0.8mM IPTG.
  
  
  

  Fig. 11 Results of the CFU Assay, plotted against induction time

  It is visually discovered that the number of colonies carrying pXMJ19-ndoA fell off in the presence of IPTG, while the same bacteria grew well without induction within the first four hours and then its CFU decrease probably due to the leaky expression. We can also see that the growth condition of the bacteria carrying the empty vector hasn’t been affected by 0.8mM IPTG, since its CFU curve increases as normal. We repeated this experiments for 3 times and all the curves show similar trends.

• Re-Learn: We can finally come to the conclusion that the ndoA does have a killing effect on C. glutamicum, and can be used in our project as the toxin gene. In the future, we plan to change the tac promoter into a more strictly inducible one for a better characterization without leaky expression.

BBa_K3796204: P-atp2 (alkali inducible promoter)

• Aim: Patp2 is an alkali-induced promoterin our composite kill switch. We aim to verify that this promoter can respond to pH stress in Corynebacterium glutamicum and we also want to document its response towards different alkalinity.
• Design: We want to test the strength of the promoter by reporter genes as well. Here we choose to use GFP because it is more stable to pH changes. The genetic circuit we used as a testing device is BBa_K3796207. Particularly, we destroyed the tac promoter on the plasmid pXMJ19 to get rid of its influence this time. Ideally, the fluorescence intensity/OD₆₀₀ should be higher in relatively high pH.
  
  
  

  Fig. 12 Genetic circuit for Patp2 verification

• Build: We used ClonExpress II one-step cloning kit (Vazyme Biotech, China) to build the circuit for testing. The expression vector was transformed into E. coli DH5α first and then into C. glutamicum by electroporation. Electrophoresis showed that we had built the circuit successfully.
  
  
  

  Fig.13 The agarose gel electrophoresis of PCR product of pXMJ19-Patp2-gfp transformed E. coil
  Lane: MK: Marker; Lane1: colony PCR product. Target: Patp2-gfp (825bp)

  
  
  
  

  Fig. 14 The agarose gel electrophoresis of PCR product of pXMJ19-Patp2-gfp transformed Corynebacterium glutamicum
  Lane: MK: Marker; Lane1: colony PCR product. Target: Patp2-gfp (825bp)

• Test: We continued to use the fluorescence intensity/OD₆₀₀ as a measurement data. After cultivating the bacteria in the LB liquid medium at 100 rpm, 30℃ for 12h, the OD₆₀₀ of the two bacteria were adjusted to nearly the same (nearly 2.0) and inoculated in the pH gradient LB liquid medium. After a cultivation of 24h, the bacteria solution was collected and washed with PBS. Then we measured its fluorescence intensity by HITACHI F-7000 according to the excitation light of 488nm and emission light of 507nm and OD₆₀₀.
  
  

  Fig. 15 Fluorescence intensity results in the verification of Patp2
  (the excitation light of 488nm and emission light of 507nm)

  Through two-way ANOVA, we can know that there’s significant difference between the control group and experiment group under different pH conditions. The relative fluorescence intensity of the control group is relatively stable at the pH range of 7-9.5, while there is a significant increase of relative fluorescence intensity in experiment group. Data shows that the difference of relative fluorescence intensity between the two groups remains high between pH=8.5 and pH=9.0, and reaches its peak near pH=9.0, indicating that the promoter Patp2 has the most ability to enhance its downstream gene expression in this pH range. Also, we can see a sharp drop of the relative fluorescence intensity in the experiment group when the pH is more than 9.5. We assume that this is because ,C. glutamicum can’t tolerate such a high alkalinity stress and the OD₆₀₀ decreases a lot, making the relative fluorescence intensity seems abnormal or irregular. Three parallel experiments were done later, which supported our opinions.

• Learn: Patp2 can respond to pH changes in C. glutamicum, and high alkalinity can improve the expression of the downstream genes of Patp2. According to our data, the peak occurs near pH=9.0, which happens to fall in the range of the alkalinity of saline-alkaline soil. The strength pf Patp2 under higher pH needs to be further tested.