Updated on 2021-11-18: You might found this file, which we prepared for the Judging Session, helpful to understand our goals, approaches and what have been achieved.

# Proof of the availability of primers and the DNA

First, to affirm the system could work properly, we started from the very beginning by testing the availability of our primers and the DNA extracted from Candida albicans by our instructor following safety rules. As Figure 1 indicates, we got positive results with both agarose gel electrophoresis and LFA.

Figure 1. positive and specific test of LAMP combined with LFA. M: DL2000 marker; N:negative control (replacing DNA of C. albicans with *S. cerevisia); C:*positive amplification of the DNA of C. alibicans

# Identifying reaction conditions

Next, we tried to define the conditions to carry out the detection, including the detection limit, the minimum amount of enzyme required in the reaction, reaction time, and temperature. The reaction time available is above 70 mins. The reaction temperature ranges from 60-65 °C. The minimum amount of enzyme catalyzing LAMP is 2 U, and the detection limit of C. albicans using LAMP is 0.4 pg/μl.

Figure 2. Testing detection conditions. (a)Testing reaction time.M: marker; O ther lanes: different reaction time as indicated. (b) Testing minimum enzyme requirement to react. M: DL2000 marker; other lanes: different reaction temperature as indicated. (c) Testing reaction temperature. M: DL2000 marker, N: negative control; other lanes: the different amount of enzyme as indicated. (d) The testing detection limit of LAMP combined with LFA. M: DL2000 marker; other lanes: different template concentration as indicated.

# Construction of Bst expression system

In order to reduce the cost of our detection kit and make it more accessible in underdeveloped areas, we constructed several parts to produce low-cost bacterial lysate that can replace commercial Bst polymerase in LAMP reaction.

A composite part was designed and constructed to achieve our goals - BBa_K3790212. It was inserted into the recombinant plasmid. The constructed plasmid was transformed into E. coli Fast-T1. We picked colonies to further grow clones in liquid medium with ampicillin and Mini-prep was performed the next day. The constructed plasmid was then verified by enzyme digestion and sequencing.

Figure 3 shows that the composite part BBa_K3790212 had been successfully inserted into the plasmid.

Figure 3. Gel electrophoresis of plasmids containing K3790212 digested with NcoI and KpnI. The first lane was loaded with DNA ladder, sizes were marked on the image. All bands are about 50 ng. Lane labeled with K3790212 is the plasmid DNA without enzymatic digestion. Lane labeled with NcoI/KpnI, is digested. The arrowhead marks the predicted excised DNA fragment approximately 1800bp.

We transformed the verified plasmids into E. coli BL21 (DE3) Positive colonies were selected by ampicillin preliminarily and then verified by colony PCR (Figure 4). Correct colonies were cultivated for subsequent experiments.

Figure 4. Gel electrophoresis of colony PCR of BL21 (DE3) clones. The first lane was loaded with DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. After obtaining the correct K3790212 recombinant plasmids, we transformed them into BL21 (DE3) competent bacteria for protein expression. Positive colonies were selected by ampicillin preliminarily and then verified by colony PCR. The arrowhead marks the target approximately 1800bp.

Using a self-made Bst polymerase compatible buffer (BPCB), we obtained the bacterial lysate (Figure 5) that works well in LAMP reaction as Bst polymerase (Figure 6).

Figure 5. Compare proteins in the pellet vs. supernatant, after BPCB lysis.Lane 2 was Bst containing supernant. We add equal volume of 2x SDS sample buffer. Lane 1 was BPCB insolvable components, from equivalent pellet. As shown in the gel, BPCB solubilizes the bacteria very well, and we do not perform 4 ℃ centrifugation after seeing this gel.

Figure 6. LAMP reactions using purified Bst or self-made bacterial lysates. The first lane was loaded with DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. For the last two lanes, we used bacterial lysates from BL21 (DE3) transformed with K37900212, with or without IPTG induction. Purified Bst represents the purified enzyme.