Team:TU Kaiserslautern/Contribution

Working with iGEM teams from years past demonstrates the importance of contributing to the scientific community. Here we detail the tac-Promotor for Escherichia coli as a part of our contribution.

Testing the influence of different IPTG-Concentrations on the induction of the tac-promotor in Escherichia coli

The tac promotor we used is an established strong E. coli promotor. The promotor is a hybrid between the trp and lac UV5 promotors (BioCat GmbH). Replacing the -35 region of the lac UV5 promotor with the -35 region of the stronger trp promotor makes this hybrid way more efficient.¹ In addition to the -35 region the tac promotor also has a Pribnow box sequence to increase the efficiency.¹ The activity of the tac promotor can be repressed by a lac repressor and activated by inducing with isopropyl beta-D-thiogalactoside (IPTG), so a high regulation is possible.¹ The iGEM team iGEM12_Uppsala introduced the part BBa_K864400 to iGEM first in 2012. It was improved by Evry_Paris-Saclay in 2017 and characterized by by SHSBNU_China in 2019. Our goal was to verify this data.

We wanted to help iGEM create a big data bank of parts, because contributing to the body of information is everything in science. We worked with the tac promotor the entire time in E. coli to express the genetical code of our protein. So, we wanted to know, which influence the IPTG concentration has on the activity of the promotor. We transformed the pGEx-6P-1_baLac vector in E. coli AD494 (DE3) competent cells. For selection we used Kanamycin and Ampicillin resistance.

We induced six liquid cultures of E. coli AD494 (DE3) pGEX-6P-1_baLac with the same optical density at 600 nm with different IPTG concentrations to compare different influences on the expression of the gene for the protein BaLac (Table 1). The cells grew at 17°C for 19 h. We took a sample before we induced and after expression for 19 h. We lysed the latter to separate the pellet and the soluble fraction with SDS-PAGE (Fig. 1).

Piston	Concentration IPTG [mM]
1	0.205
2	0.375
3	0.500
4	1.00
5	1.500
6	2.500

Fig. 1: SDS-PAGE of the test expression with different IPGT concentrations.

We induced E. coli AD494 (DE) pGEX-6P-1_baLac with six different IPTG concentrations. (A) shows the IPTG concentrations 13 (1: 0,25 mM; 2: 0,375 mM; 3: 0,5 mM), (B) shows the IPTG concentrations 46 (4: 1 mM; 5: 1,5 mM; 6: 2,5 mM). Samples were taken before induction and after induction expressing for 19 h. The latter were disrupted by sonication and insoluble and soluble fraction were separated. The red boxes show the produced translation fusion protein (BaLac and GST). The western blot was detected by anti-GST-antibodies (first antibody) and anti-Goat alkaline phosphatase conjugated antibodies (second antibody). Marker: New England BioLabs ® Blue Protein Standard Broad Range.

In summary, we can say, that with increasing concentration of IPTG the production of protein increases as well (Fig. 1). That means, that there must have been a higher expression of the gene of interest (balac and gst). But you have to keep in mind, that sometimes E. coli has problems to fold foreign proteins correctly. This gets even worse if the expression of this protein is high. Figure 1 (B) shows, that the more IPTG is used, the more protein is in the insoluble fraction (pellet). That’s the reason why an IPTG concentration of 0.5 mM has established itself in our protein production – there we got the highest yield of soluble protein with the lowest concentration of IPTG.

Testing the pAR Promotor in Chlamydomonas reinhardtii in combination with laccases

The pAR-promotor has, since its introduction to the iGEM-community, been tried and tested. After all its characteristics as a fusion promotor have proven to be efficient in working with Chlamydomonas reinhardtii. It is made made from the promotors from the proteins HSP70 and RBCS2. HSP70 is a heat shock protein, therefore its promotor is slightly heat resistant.² While RBCS2 is the gene of the small subunit 2 of RuBisCO and is expressed constitutively.

As such we decided to test the efficiency of the pAR-promotor in combination with the mutated laccase from Botrytis aclada (BaLac) and the laccase from a marine organism (marLac). At first we assembled a Level 2 construct consisting of the spectinomycin-resistance, the coding sequence of the laccases (BaLac or marLac) with the pAR-promotor, a 3xHA-tag for detection and the RPL23 terminator. (as seen in the figure below)

Fig. 2: Level 2 construct of cytosolic BaLac (BBa_K3589207) and cytosolic marLac (BBa_K3589208)

It includes the constitutive pAR-promotor, the enzyme (BaLac or marLac) fused with a 3xHA tag for detection and a spectinomycin-resistance for selection.

Fig. 3: Proof for expression of a) BaLac and b) marLac in Chlamydomonas reinhardtii

It shows the Immunoblot of 12 spectinomycin-resistant colonies, which were transformed with a) construct (BBa-K3589207) and b) construct (BBa_K3589208) (as seen in Fig. x). 2 µg Chlorophyll were always loaded onto the gel. At ca. 70 kDA (indicated with an arrow) is the expression of BaLac in the transformants 5,9 and 11. At ca. 60 kDA (indicated with an arrow) can the expression of marLac in the transformants 5,9 and 11 be seen. A 3xHA-tagged protein was used as positive control, while the recipient strain (UVM4) was used as a negative control.

As can be seen in the figure above (Fig. 3) an expression of both BaLac and marLac have been achieved by using the pAR-promotor. Even though activity could not be confirmed, the pAR-promotor still proves to be efficient for expressing in C. reinhardtii and as such can be recommended to everyone working with C. reinhardtii

References

(1) de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21-5. doi: 10.1073/pnas.80.1.21. PMID: 6337371; PMCID: PMC393301.
BioCat GmbH

(2) Schroda, M.; Blöcker, D.; Beck, C. F. The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. The Plant journal : for cell and molecular biology 2000, 21 (2), 121–131. DOI: 10.1046/j.1365-313x.2000.00652.x.