In vivo selection
The use of the same pUC19-derived pMB1 origin of replication within pSB1C3 and pSB1K3 could cause problems, because E. coli is not able to control the amount of plasmid in the cell if both plasmids have the same ori. The next step was the exchang of the backbone of the part with SH2-cI and the reporter from pSB1K3 to pSB4C5. The pSB4C5 vector uses the pSC101 origin of replication. For different antibiotic resistances the coding sequence of the other fusion protein HA4-RpoZ was cloned in the pSB1K3 vector. To test the efficiency of the new constructs, both were transformed in the E. coli strain KRX together. A comparison of a culture with cells carrying both plasmids and a culture with only the pSB4C5 plasmid containing the reporter and the SH2-cI fusion protein reveals visible differences in the RFP fluorescence intensity. The culture with both plasmids has a higher visual red color. To validate the results, the two cultures were measured in the Tecan plate reader (figure 1).
![](https://static.igem.org/mediawiki/2016/1/13/Bielefeld_CeBiTec_2016_10_18_SEL_RFP_B2H.png)
Although it is shown that the bacterial two-hybrid system is working, the output signal is lower than expected. Two possibilities could increase the transcription activity of the bacterial two-hybrid selection system. The first opportunity is the reduction of the background activity of the promoter. The used promoter BBa_K2082210 is a little bit leaky. Therefore, without any interaction of the two fusion proteins the gene is a little bit expressed. In the measurement the reporter was brought in the pSB4C5 plasmid. For the plasmid itself a very high amount of DNA was measured after every plasmid isolation. Therefore, we expect it to be a high copy plasmid. Although the promoter is only a little bit leaky, the high amount of plasmid leads to a high number of the reporter gene transcripts. And if every reporter is a little bit leaky, the sole number of the reporter causes a high signal of RFP. This is seen in the figure X, considering that the RFP signal is extremly high, without any transcription activation through the two fusion proteins. The second possibility for the low activation rate of the selection system is the native rpoZ gene in the E. coli cell. RpoZ is a subunit of the RNA polymerase I core complex. Under normal conditions the RpoZ protein would be guided by the beta´-subunit to the core complex of the polymerase as the last unit. If E. coli produces a lot of native RpoZ it competes with the RpoZ coupled with the HA4 Monobody. Therefore, it is possible, that most of the most polymerases build their core with the native RpoZ instead the RpoZ-HA4 fusion protein. In this complex it is not able to be recruited by the second fusion protein and the transcription activation of the reporter stay off.
An interesting fact is, that the RNA polymerase do not necessarily needs the RpoZ protein of their functionality (Gosh et al., 2001; Mathew et al., 2005). Therefore, the next step for the bacterial two-hybrid system is a knock-out of the native rpoZ gene combined with a knock-in of the beta-lactamase containing reporter(BBa_K2082238) in the genome of our working strain JS200 of E. coli. With this attempt it is possible to decrease the background activity of the reporter by only one copy of the reporter left in the cell and to eliminate the expression of the native RpoZ and preventing the competiton of the native RpoZ with the RpoZ-HA4 fusion protein in one step.
![](https://static.igem.org/mediawiki/2016/c/c7/Bielefeld_CeBiTec_2016_10_18_SEL_Cas_Illustration.png)
Some bands on the gel are 2,300 to 2,400 bp in size. This is the expected length of the genomic sequence of the flanked rpoZ gene. Therefore, these results are negative. However, five bands on the gel are longer with about 3000 bp. With a length of about 1000 bp of the reporter and each time 1000 base-pairs of the two flanked fragments of the designed DNA construct, the expected length for a successful integration of the reporter gene in the genome was given. Therefore, the knock-out, knock-in experiment was successful and the reporter was perfectly integrated into the JS200 genome.
![](https://static.igem.org/mediawiki/2016/3/3d/Bielefeld_CeBiTec_2016_10_19_SEL_CRISPR.png)
Although the integration was successful, it was not possible to test the advantages of this modification in vivo, due to the time limitations. The different HA4 mutants could not be tested, because the cloning from pSB1C3 to pSB1K3 took long. Unfortunately the final measurement of the system with the mutants could not be completed. Nevertheless, we can proudly report the, that we designed a functional bacterial two-hybrid system and we paved the way for upcoming experiments with the system.