Team:IISER Berhampur/Results

iGEM IISER_Berhampur

Experimental Results

As the global pandemic continues, we couldn’t get hold of our laboratories on time. However, we got access to our labs during the last couple of months before wiki-freeze. Although we ordered the reagents beforehand, we couldn’t get them all on time due to transportation and other issues. With the available reagents, we did some basic and optimization experiments. We have attached below the results of the experiments done so far.

BAC Clones plasmid isolation

Mycobacterium tuberculosis (a kind gift from Prof. Rajesh Gokhale, National Institute of Immunology, Delhi) was isolated with the help of the NEB Monarch Plasmid isolation kit (New England Biolabs). We did an agarose gel run on those BAC plasmids in a 0.8% agarose gel with 10kb Ladder (1kb minimum) and got the following result. Then we did an in-silico agarose gel run for rpoB and katG genes as a preliminary analysis for PCR to stock the gene fragments (Lane 1 - rpoB, Lane - katG). For the Protocol, refer to the Experiment page.

Results are as follows:

BAC clone plasmid isolation

In silico Gel Run

The absorbance of purified recombinant venus protein :

For the Preliminary analysis of hardware to detect fluorescence, we measured the absorbance of purified Venus protein with emission excitation (YFP: 505 nm, EGFP: 488). It has properties similar to EGFP to perform an optimization experiment for our detection system.

Absorbance of venus :

A = εcl (“Beer-Lambert Law”)
From this equation, we can calculate the concentration present in well 12
c = A/εl
L = 1cm
ε = 92,200M^-1L^-1 (Excitation coefficient of venus)
A = 1.23 (plate reader)

c = A/εl
c = 13.3μM

Clear plates used for Absorbance

Absorbance of venus protein (3D Graph)

Absorbance of venus protein

Fluorescence of purified recombinant venus protein :

For the chemical kinetics of our experiment modeling, we analyzed the fluorescence emission of venus protein in fluorometry (YFP: 520, EGFP: 509). This helped us in the optimization of EGFP fluorescence emission which we will be using in our detection kit.

The 7th well showed a S/N (Signal to Noise ratio) ≈ 10³ during the fluorescence check.
Since the dilution is halved in the next well from the well 12,

The concentration of 7th well = Absorbance / dilution factor = 13.3/2⁵ = 0.4μM

In moles, 0.4μM = 40 picomoles.
Hence, a minimum concentration of 0.4μM of venus protein is required for a fluorescence S/N of ≈ 10³.

Fluorescence of venus protein (3D Graph)

Fluorescence of venus protein

Fluorescence spectrum of venus protein

NCBI Basic Local Alignment Search Tool (BLAST)

NCBI BLAST is one of the well-known tools used for nucleotide or protein sequence alignment. In order to make our sgRNAs with minimal off-target effects, the spacer sequences which we had identified from Mycobacterium tuberculosis H37Rv (complete genome) from the NCBI database (NCBI Reference Sequence: NC_000962.3) were run through the NCBI algorithm to assess their specificity. Here, BLAST serves the purpose of finding any sequences which are identical or similar to the spacer region in sgRNA.

Shown below are the results obtained from Multiple Sequence Alignment Viewer (MSA) version 1.20.1 on NCBI BLAST. The top 20 similar alignments for each spacer sequence are shown here. The 20 bases in the spacer sequence are colour coded.

Results for katG (wild type) spacer sequence

Results for katG (Mutant) spacer sequence

Results for rpoB (wild type) spacer sequence

Results for rpoB (mutant) spacer sequence

Standardization of Plasmids

Since we did not have lab access for most of the iGEM 2021 period and our access to lab materials got delayed due to the lockdown restrictions, we planned to optimize our approach for cloning and transformation experiment protocols for our CODE M Constructs using in-silico RE digestion and gel electrophoresis.

pLBH559_Tet-HisCas14a1Locus is a plasmid that expresses 10xHis-Cas14a1 locus (including crRNA and tracrRNA) under control of a tetracycline-inducible promoter. It has a pTet vector backbone and it consists of a coding region for Chloramphenicol acetyltransferase (CmR) and p15A origin. It was added to addgene by Jennifer Doudna (Addgene plasmid # 112502 ;http://n2t.net/addgene:112502 ; RRID:Addgene_112502).

In-silico Restriction Digests

In-silico restriction digests were performed in pLBH559_Tet-HisCas14a1Locus to identify the restriction sites to be used for excision of 6x-His Tagged Cas14a1 along with TEV protease (which is shown as ORF a in the restriction digestion map given below) for cloning them into pSB1C3 and also, to avoid any illegal restriction sites. We used the NEB cutter tool http://nc2.neb.com/NEBcutter2/ for this purpose.

Caption: Restriction Enzyme Digestion Map for pLBH559_Tet-HisCas14a1Locus; for all commercially available NEB enzymes.

We double digested it with EcoR1 and Pst1 enzymes. We found that EcoR1 and Pst1 sites were present in the ORF a, as shown in the figure below, which is the coding region for 6x-His Tag, Cas14a1, and TEV protease. Also, in another double digestion using Xba1 and Spe1, the sites were not present in ORF a. So, we propose to avoid using EcoR1 and Pst1 restriction enzymes and instead use Xba1 and Spe1 enzymes for cloning protocols that will be using this plasmid. We did a virtual gel run to get an expected gel image to be matched with an experimental gel run. We set a parameters of 1% agarose and 1kb DNA ladder as marker.

Caption: In-silico custom double digests for pLBH559_Tet-HisCas14a1Locus done using: A) EcoR1 and Pst1, and B) Xba1 and Spe1 restriction enzyme sets.

EcoR1 and Pst1 double digest

Xba1 and Spe1 double digest

Caption: Virtual gel runs using the enzyme sets A and B.

Gel run for EcoR1 and Pst1

gel run for Xba1 and Spe1