Team:Hong Kong HKUST/pneumosensor/results

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<i>comE</i> sequence contained an illegal SpeI site, so we designed a set of overlapping primers for site-directed mutagenesis:
<i>comE</i> sequence contained an illegal SpeI site, so we designed a set of overlapping primers for site-directed mutagenesis:
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<br>
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<i>Mutagenesis forward primer: CGCTATTATCGTCTTTATCACTAGCCGATCAGAGTTTGCGACTCTAAC
+
Mutagenesis forward primer: CGCTATTATCGTCTTTATCACTAGCCGATCAGAGTTTGCGACTCTAAC
<br>
<br>
-
<i>Mutagenesis reverse primer: GTTAGAGTCGCAAACTCTGATCGGCTAGTGATAAAGACGATAATAGCG
+
Mutagenesis reverse primer: GTTAGAGTCGCAAACTCTGATCGGCTAGTGATAAAGACGATAATAGCG
<br><br>
<br><br>
However, site-directed mutagenesis attempts were unsuccessful, so the gene was extracted in two parts using (i) <i>comE</i> forward primer & mutagenesis  
However, site-directed mutagenesis attempts were unsuccessful, so the gene was extracted in two parts using (i) <i>comE</i> forward primer & mutagenesis  

Revision as of 14:01, 17 October 2014




Pneumosensor Results





Detection Module


Overview

The two-component regulatory system in S. pneumoniae, consisting of the receptor ComD and its response regulator ComE was to be used in detecting the autoinducer molecule, competence-stimulating peptide (CSP) and so detect S. pneumoniae populations correspondingly. The activity of the comCDE operon promoter (PcomCDE) is induced by phosphorylated ComE. In order to facilitate characterization of PcomCDE, we use the phosphorylmimetic ComE mutant, ComED58E, in the pKHS plasmid which was kindly sent to us by Martin et al., from the Université de Toulouse. The characterization of PcomCDE is for the purpose of linkage to the σx promoters module by regulating expression of the sigma factor.

Construct

Bacterial Strain
The bacterial strain of E. coli used was DH10B.

comD and comE gene
comD gene and comE gene were cloned from NCTC 7465 S.pneumoniae strain genomic DNA by PCR using Phusion Polymerase.



ComD Tag Protein



We engineered in a FLAG protein tag in the 3’ end of ComD by including the sequence in comD extraction primer.

comD forward primer:
TCTGGAGAATTCGCGGCCGCTTCTAGATGGATTTATTTGGATTTGGGACGG
[6’cap][20’ RFC10 prefix][25’ Streptoccocus pneumoniae/NCTC7465/comD]

comD reverse primer with FLAG tag:
GCCGGACTGCAGCGGCCGCTACTAGTATTATTACTTGTCGTCATCGTCTTTGTAGTCTCATTCAAATTCCCTCTTAAATCTAATGAT
[6’ cap][21’ RFC10 suffix][6’ reverse complement stop codon][25’ reverse complement FLAG protein ][30 reverse complement Streptoccocus pneumoniae/NCTC7465/comD]

comD sequence contained two illegal EcoRI sites, so we designed a set of partially overlapping primers for site-directed mutagenesis:
1st site Mutagenesis forward primer: GACCTTCTTTGCAATGAACTCTCATAAAG
1st site Mutagenesis reverse primer: CCATTTGAACTTTATGAGAGTTCATTGC
2nd site Mutagenesis forward primer: GAAACAGTTATTGTGATTCAAAATTCATG
2nd site Mutagenesis reverse primer: CGTCATTTTACATGAATTTTGAATCAC

However, site-directed mutagenesis attempts were unsuccessful, so the gene was extracted in three parts using (i) comD forward primer & 1st site mutagenesis reverse primer; (ii) 1st site forward primer & 2nd site reverse primer; (iii) comD reverse primer & 2nd site mutagenesis forward primer. The two fragments were then ligated with the pSB1C3 backbone through Gibson Assembly.



ComE Protein


comE was extracted from pKHS-come kindly sent to us by Dr. Don Morrison (Université de Toulouse, UPS, Laboratoire de Microbiologie et Génétique Moléculaires). Extraction was done using the following primers:

comE forward primer:
TCTGGAGAATTCGCGGCCGCTTCTAGATGAAAGTTTTAATTTTAGAAGATG
[6’ cap][20’ RFC10 prefix][25’ Streptoccocus pneumoniae/NCTC7465/comE]

comE reverse primer:
GCCGGACTGCAGCGGCCGCTACTAGTATCACTTTTGAGATTTTTTCTCTAA
[6’ cap][21’ RFC10 suffix][24’reverse complement Streptoccocus pneumoniae/NCTC7465/comE]

comE sequence contained an illegal SpeI site, so we designed a set of overlapping primers for site-directed mutagenesis:
Mutagenesis forward primer: CGCTATTATCGTCTTTATCACTAGCCGATCAGAGTTTGCGACTCTAAC
Mutagenesis reverse primer: GTTAGAGTCGCAAACTCTGATCGGCTAGTGATAAAGACGATAATAGCG

However, site-directed mutagenesis attempts were unsuccessful, so the gene was extracted in two parts using (i) comE forward primer & mutagenesis reverse primer; (ii) comE reverse primer & mutagenesis forward primer. The two fragments were then ligated with the pSB1C3 backbone through Gibson Assembly.

PcomCDE


The promoter region of the comCDE operon only contains 67bp. By estimating the position of the transcription start site of PcomCDE, the total size of the promoter region should be 89bp. We obtain the promoter region by oligo annealing. In designing the forward and reverse oligos, we added the XbaI cut site to the prefix and SpeI cut site to the suffix at the two ends of the oligos respectively. The backbone pSB1C3 was used. PcomCDE was ligated with GFP generator (BBa_E0240), which contains a RBS (BBa_B0032), GFP (BBa_E0040) and double terminator (BBa_B0015). The purpose of this construct is to measure the functionality of PcomCDE in the presence and absence of phosphorylated ComE by green fluorescence. BBa_E0240 was obtained from 2014 iGEM distribution kit. The bacterial strain of E.coli used was DH10B. We have tried to ligate PcomCDE with BBa_E0240, but unfortunately due to time limitation, we were not able to verify the sequence of our ligated products.

PcomCDE forward oligo:
CTAGAGAAAAAGTACACTTTGGGAGAAAAAAATGACAGTTGAGAGAATTTTATCTAAAACGAAATTCCATTTTGTATAATGGTTTTTGTAA
GTTATA
[6'XbaI prefix][89'PcomCDEregion][2'SpeI suffix]

PcomCDE reverse oligo:
CTAGTATAACTTACAAAAACCATTATACAAAATGGAATTTCGTTTTAGATAAAATTCTCTCAACTGTCATTTTTTTCTCCCAAAGTGTACTTT
TTCT
[6'SpeI suffix][89'PcomCDEregion][2'XbaI prefix]

ComED58E


The phosphorylmimetic comE mutant, comED58E was sent by Martin et al., the pKHS plasmid. pKHS is an expression vector, which contains a T7 promoter and kanamycin resistance gene. T7 promoter is induced by isopropyl β-D-1-thiogalactopyranoside (IPTG).




S. pneumoniae σx Promoters Module


Overview

The activity of Com-Box promoter is turned on by a specific sigma factor that is produced by a regulatory gene comX. The σx will bind to the Com-Box promoter region and activate gene expression. σx serve as an inducer with high specificity as it binds to an area of several specific 8 base pairs (TACGAATA) on the Com-Box promoter. This σx-Com-Box system could be used as a highly specific reporting system in our S.pneumonia detection platform. However in nature, ComX protein will be degraded by ClpXP enzyme which exists in E. coli and some other bacteria. Hence, to ensure the induction of Com-Box promoter by σx, ComW protein is needed as it functions to protect σx from being degraded by ClpXP. ComW protein will be degraded instead, increasing the amount of σx produced.

Construct
The three main components of the construct are comX gene, comW gene, and Com-Box promoter. We assembled comX and Com-Box promoter in one vector plasmid, while comW in a different plasmid. The system will be fused with a tagging protein and a reporting protein. Tagging protein is essential for detecting the σx and ComW protein expression by means of western blot. Reporting protein which is fluorescence protein is needed for reporting purpose, hence σx-Com-Box system could serve as a specific reporting system that will be useful for many synthetic constructs. σx generator and Com-Box promoter construct will be assembled separately in different plasmid before being combined into one plasmid.

σx Generator construct (BBa_K1379006) and comW construct

Backbone pSB1C3 was used for σx generator construct and comW construct. comX gene / comW gene were fused with BBa_K880005 which contains a constitutive promoter (BBa_J23100) and strong RBS (BBa_B0034). The purpose of this strong constitutive promoter and strong RBS is to unsure the large production of σx and ComW protein throughout time. Then, a double terminator (BBa_B0015) is fused with the promoter, RBS, and comX. BBa_K880005 and BBa_B0015 were obtained from 2014 iGEM distribution kit.

Construct using pSB1C3 backbone consisting only σx CDS (BBa_K1379004), and σx followed by double terminator BBa_B0015(BBa_K1379045) was also built to facilitate assembly of σx to promoters and RBSs of choice.

Bacterial Strain
The bacterial strain of E. coli used was DH10B. Since this strain of E. coli has clpXP degradation enzyme which targets ComX for degradation, an excess amount of ComX protein is required to maintain enough amount of ComX for Com-Box promoter induction.

comX and comW gene
comX gene and comW gene were cloned from NCTC 7465 S.pneumoniae strain genomic DNA by PCR using Vent Polymerase.


ComX Tag Protein


We engineered a C-myc protein tag in the 3’ ends of comX by including the sequence in comX extraction primer.

3’ primer to extract comX with engineered C-myc tag gene sequence:

GCCGGA CTGCAGCGGCCGCTACTAGTA TTATTA CAGATCCTCTTCTGAGATGAGTTTTTGTTC GTGGGTACGGATAGTAAACTCCTTAAACAC

[6’ Cap] [21’ SpeI and PstI restriction site] [6’ terminator sequence] [30’ C-myc protein] [30' reverse complementary of 3’ comX]

ComW Tag Protein


We engineered a FLAG protein tag in the 3’ ends of comW by including the sequence in comW extraction primer.

3’ primer to extract comW with engineered FLAG tag gene sequence:

GCCGGA CTGCAGCGGCCGCTACTAGTA TTATTA CTTGTCGTCATCGTCTTTGTAGTC ACAAGAAATAAAACCCCGATTCATTACCAATT

[6’ Cap] [21’ SpeI and PstI restriction site] [6’ terminator sequence] [24’ FLAG protein] [32' reverse complementary of 3’ comW]

PcelA (BBa_ K1379002) and PcomFA (BBa_ K1379003) construct

Backbone pSB1C3 was used for PcelA and PcomFA construct. PcelA / PcomFA gene was fused with BBa_E0240, which contains a medium RBS (BBa_B0034), GFP (BBa_E0040) and double terminator (BBa_B0015). The purpose of this GFP generator is to indicate the functionality of PcelA and PcomFA in the presence and absence of σx. BBa_E0240 was obtained from 2014 iGEM distribution kit. The bacterial strain of E. coli used is DH10B.

PCelA / PcomFA gene



Identifying the Possible Promoter Regions
To date (12 Oct 2014), the core/minimal promoter region of PcelA has not yet been experimentally defined. In locating the promoter region required to initiate transcription, iGEM 2014 Hong_Kong_HKUST team attempted in making educated guesses based on relevant information available from the literature. The Com-Box promoter consensus sequence “TACGAATA” was BLASTed for targets in the Streptococcus pneumoniae genomes of strains R6, D39, ATCC7699 and NCTC7465 in the NCBI database. Genome of strain NCTC7465 was particularly given attention because its gDNA was available for manipulation. A list of loci with annotated genes returned. σx was known to turn on late competence gene and therefore loci containing any of those genes documented were favored and filtered for. Those loci were then manually checked for consensus among the 4 genomes mentioned above. A sequence of 67 base pairs stood out as a promising target because it was 1) upstream of a late competence genes celA (encodes competence protein CelA), and 2) was identical across the 4 genomes. This 67 bp region has varying upstream sequences and the potential promoter region can reach as far as 200bp. Different truncations (67, 100, 150, 180, 249, 300 bp) were planned for deciding the minimal promoter region, but in the course of construction, only the 100bp version could be finished in time. It was tested to be functional and therefore submitted as PcelA.

PcelA and PcomFA gene were both cloned from the genomic DNA of S. pneumoniae strain NCTC7465 by PCR using Vent Polymerase. The difference between these two promoters is the whole sequence of PcomFA was obtained from Wellcome Trust Sanger Institute, a British genomics and genetics research institute. (https://www.sanger.ac.uk/)

PcelA Forward primer: TTTCTGTCTAGAGTTGACCAAGGAAGACTATTTTGC

PcelA Reverse primer: GCCGGACTGCAGCGGCCGCTACTAGTAATTTTCTCCTCTCTTAGATTATTCGTAAGAGG

PcomFA Forward primer: TTTCTGTCTAGAGTGGACTTGGCCGTCCTCT

PcomFA Reverse primer: GCCGGACTGCAGCGGCCGCTACTAGTAGACGTTCTTCTTCTGTTAATTCATTCTCAG

Assembly and Characterization

Assembly
comX and comW construct contain 3 parts that need to be assembled: BBa_K880005 which contains constitutive promoter and RBS, comX engineered with C-myc tag / comW engineered with FLAG tag, and a double terminator in pSB1C3 backbone. Promoter, RBS, comX engineered with C-myc tag, and double terminator were combined using traditional digestion and ligation method. The ligation product was confirmed by digestion check and sequencing.

Com-Box construct also contains 3 parts that need to be assembled: PcelA/PcomFA promoter, BBa_E0240 which contains RBS, GFP and double terminator, and pSB1C3 backbone. All three parts were combined using traditional digestion and ligation method. The final ligation product was confirmed by digestion check and sequencing.

Characterization
RPU (Relative promoter unit) and leakage will be measured as a characterization of 100 base pairs Com-Box promoter (PcelA), and 160 base pairs Com-Box promoter (PcomFA). For Com-Box promoter characterization, σx generator construct which contains BBa_K880005, comX gene, and BBa_B0015, is ligated with PcelA / PcomFA construct containing Com-Box promoter and GFP generator. In order to characterize the two Com-Box promoters, σx generator-Com-Box construct was migrated from pSB1C3 to pSB3K3. RPU are measured with BBa_J23101 Andersen family promoter as a reference promoter.


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